International Immunology Advance Access originally published online on February 16, 2007
International Immunology 2007 19(4):365-373; doi:10.1093/intimm/dxm002
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Follicular exclusion of autoreactive B cells requires Fc
RIIb
1 Department of Pediatrics, Harvard Medical School, Boston, MA, USA
2 Division of Pediatric Nephrology, Massachusetts General Hospital, Boston, MA, USA
3 Department of Pathology, Harvard Medical School, Boston, MA, USA
4 CBR Institute for Biomedical Research, Boston, MA, USA
Correspondence to: E. Paul; E-mail: epaul{at}partners.org
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Abstract |
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In non-autoimmune mice, the 3H9 transgenic Ig heavy chain can pair with endogenous Ig
1 light chains to generate B cells with specificity for DNA. These autoreactive cells are actively regulated in vivo, as indicated by the exclusion of 1 cells from the splenic B cell follicle and the absence of auto-antibody production. To study the role of Fc
receptor IIb (FcRIIb) in peripheral B cell tolerance, FcRIIb–/– mice were crossed with C57BL/6 mice bearing a site-directed knock-in of the 3H9 transgene. 3H9FcRIIb–/– mice become autoreactive, lose the follicular exclusion of anti-DNA B cells and instead have 1 B cells located within splenic germinal centers. They have increased frequencies of splenic auto-antibody-producing cells and elevated titers of IgG anti-DNA auto-antibody. The data implicate an FcRIIb-dependent checkpoint that can exclude autoreactive B cells from splenic follicles. By restricting their participation in germinal center reactions, this putative checkpoint helps attenuate the production of potentially pathogenic auto-antibodies. The data further suggest that this FcRIIb-dependent regulation is B cell autonomous.
Keywords: autoimmunity, Fc receptors, lupus, rodent, tolerance
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Introduction |
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In healthy individuals, the development of autoreactive B cells is strictly regulated by mechanisms including receptor editing, clonal deletion and anergy. Breakdown in one or more of these tolerogenic mechanisms can result in development of auto-antibodies and autoimmune disease. Systemic lupus erythematosus (SLE) is one such progressive disease, characterized by self-reactive antibodies against DNA and other nuclear antigens, that may be caused in part by defects in B cell tolerance [reviewed in (1)].
Numerous Ig transgenic mouse models have been developed to study the regulation of B cells that produce auto-antibodies against the nuclear auto-antigens characteristic of SLE [reviewed in (2)]. One well-described model uses the 3H9 Ig heavy chain isolated from an anti-DNA B cell of an MRLlpr/lpr mouse that was ill with lupus-like disease (3–5). The 3H9 heavy chain generates an anti-DNA autospecificity when paired with various kappa or lambda light chains including Ig1 (6–8). In conventional 3H9 transgenic mice (3H9-Tg), the transgenic construct encodes the 3H9 VDJ segments fused to the BALB/c IgMa constant region and is randomly inserted into the mouse genome (4). In 3H9-Tg mice, the 3H9-IgM heavy chain pairs with endogenous 1 light chains to generate B cells with specificity for DNA at a frequency that allows investigators to identify a subset of autoreactive B cells simply by virtue of the light-chain allotype they express (9).
3H9-Tg BALB/c mice remain tolerant despite the existence of a relatively large population of autoreactive 3H9 1 splenic B cells (9, 10). These anti-DNA B cells accumulate at the T–B interface of the splenic follicle and are evidently prevented from progressing into germinal center reactions. The follicular exclusion of these autoreactive cells correlates with their functional anergy both in vivo and in vitro (9–11). Moreover, the presence or absence of follicular exclusion correlates with the presence or absence of B cell tolerance in many (12, 13) but not all (14) experimentally manipulated 3H9-Tg animals. In most cases, loss of tolerance correlates with loss of follicular exclusion, suggesting that follicular exclusion represents a peripheral tolerogenic checkpoint of B cell regulation. Nevertheless, follicular exclusion remains a relatively enigmatic manifestation of a peripheral tolerogenic checkpoint as only limited details of the cellular and molecular mechanisms responsible for follicular exclusion are available (13, 15).
Excluded autoreactive B cells display reduced levels of cell-surface B cell receptor (BCR) and CD21/35. This phenotype is consistent with previous exposure to antigen and subsequent maturational arrest, perhaps in absence of a second stimulatory signal such as T cell help (9, 13, 16). Alternatively, the excluded cells may be actively rather than passively arrested by an inhibitory stimulus. Fc receptors, expressed by numerous cells in both the innate and adaptive immune systems, are implicated in modifying the severity of SLE and lupus-like disease (17–19). Among the Fc receptors which bind IgG, the Fc receptor IIb (FcRIIb), expressed primarily on B cells and myeloid cells, is a low-affinity receptor for IgG that transmits an inhibitory signal via its cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) domain [reviewed in (19–21)]. When IgG-containing immune complexes cross-link FcRIIb, the negative stimulation interrupts and/or prevents activation of the cell in question. Bolland and Ravetch (22) were the first to demonstrate the importance of this molecule in B cell tolerance. C57BL/6 (B6) animals without FcRIIb develop a progressive lupus-like autoimmune disease characterized by B cell hyperactivity. The mechanisms by which FcRIIb deficiency allows for the emergence of pathogenic autoimmunity remains to be elucidated.
3H9 animals deficient in FcRIIb were generated to test the hypothesis that follicular exclusion depends on normal FcRIIb signaling. If FcRIIb participates in follicular exclusion and peripheral B cell tolerance, then 3H9FcRIIb–/– mice are predicted to lose follicular exclusion and to become autoreactive. A 3H9 knock-in strain (instead of the conventional 3H9-Tg mice) was used to test this prediction. These 3H9 mice bear a site-directed insertion of the 3H9 VDJ transgene into the endogenous IgH locus (23). Once back-crossed onto the B6 genetic background, 3H9FcRIIb–/– mice reveal for the first time that FcRIIb participates in the peripheral regulation of anti-DNA B cells in the 3H9 model of B cell tolerance.
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Methods |
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Mice
BALB/c mice with a site-directed insertion of the 3H9 heavy-chain transgene (23) were obtained from M. Weigert (University of Chicago, Chicago, IL, USA) and back-crossed 10 generations to B6 animals. Fc
RIIb–/– mice on the B6 background (22) were obtained from J. Ravetch (Rockefeller University, New York, NY, USA). B6 and RAG1–/– mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Mice were housed in the specific pathogen-free animal facility of CBR Institute and handled in accordance with Institutional Animal Care and Use Committee guidelines. Only female mice were used in the experiments described below.
Bone marrow chimeras
Bone marrow (BM) chimeras were made by intravenous injection of BM (20 x 106 cells isolated from long bones of 3H9FcRIIb+/+ or 3H9FcRIIb–/– animals) into B6 female mice that were lethally irradiated with two doses of 650 rads separated by 6 h. Mixed BM chimeras were made by co-transfer of 3H9FcRIIb–/– marrow and RAG1–/– marrow into lethally irradiated B6 female recipients at a ratio of 1:10. Donors were 8- to 12-weeks old and recipients were 5- to 8-weeks old. Chimeric animals were studied at 3 and 9 months after BM transfer.
Flow cytometry
Splenic single-cell suspensions were stained for FACS analysis with the following reagents from BD PharMingen (San Jose, CA, USA): FITC-conjugated anti-B220, anti-CD23 and anti-IgD; PE-conjugated HSA and anti-CD138; biotinylated anti-1 and Cy5-conjugated anti-CD21. The 331.12 anti-IgM monoclonal (obtained from K. Hayakawa, Philadelphia, PA, USA) was purified from hybridoma cell supernatant and conjugated with Cy5 (Molecular Probes, Eugene, OR, USA) according to manufacturer's guidelines. Biotinylated reagents were detected with allophycocyanin-conjugated streptavidin (BD PharMingen). Four-color FACS data acquired on a FACSCalibur were analyzed using CellQuestPro software (BD Biosciences, San Jose, CA, USA).
Immunohistology
Frozen spleen sections, embedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA, USA) and fixed in acetone for 3 min at 4°C, were blocked with anti-CD16/32 (BD PharMingen) in 1% BSA/0.1% Tween 20/PBS. Staining reagents for confocal and light microscopy included FITC-conjugated peanut agglutinin (PNA), biotinylated anti-1, PE-conjugated anti-B220, APC-conjugated streptavidin, phosphatase-conjugated streptavidin (all from BD PharMingen) and peroxidase-conjugated anti-FITC (Boehringer Mannheim, Germany). Enzyme-linked reagents were detected with the substrates 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO, USA) and 1-Step TM NBT/BCIP (Pierce, Rockford, IL, USA).
DNA ELISA
Ninety-six-well microtiter plates (Immulon 2HB, Thermo Electron Corp., Milford, MA, USA) were coated overnight with 100 µg ml–1 sonicated DNA (Eppendorf North America Inc., Westbury, NY, USA) in Tris–EDTA (TE) buffer. After washing with 0.1% Tween 20/PBS, plates were blocked with 5% BSA/PBS. Sera diluted 1:100 were added and incubated for 1 h at 37°C. Plates were washed and bound antibodies were detected with phosphatase-conjugated goat anti-mouse IgM or IgG and p-nitrophenyl phosphate substrate (Sigma).
DNA enzyme-linked immunosorbent spot
Twenty-four-well tissue cell culture plates (Costar, Corning, NY, USA) were coated overnight with 300 µl of 100 µg ml–1 DNA in TE. After washing with PBS, plates were blocked with 1% BSA/PBS and then cells were added in serial dilutions from 106 to 103 cells/ml in RPMI media supplemented with 2% FCS and were incubated overnight at 37°C. After washing with 0.1% BSA/PBS, phosphatase-conjugated goat anti-mouse IgM or IgG (Sigma) was applied for 2 h at 37°C. Plates were washed and developed by adding 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Roche Diagnostics Inc., Indianapolis, IN, USA) at 1 mg ml–1 in adenosine monophosphate buffer containing 0.6% agarose. Spots were counted manually.
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Results |
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Increased frequency and activity of autoreactive B cells in absence of Fc
RIIb3H9 mice are B6 animals with a site-directed VDJ knock-in of the 3H9 transgene (23). The 3H9 Ig heavy chain can pair with endogenous
1 light chains to generate autoreactive BCRs (9). To investigate the role of FcRIIB in the regulation of autoreactive 3H9 1 B cells, FACS analysis was used to measure their frequency among splenic B cells (Fig. 1). Splenocytes stained for B220, Ig1, IgMa (transgenic allotype) and IgMb (endogenous allotype) revealed that the frequency of 1 B cells is increased in spleens of 3H9FcRIIB+/+ animals compared with spleens of non-transgenic wild-type (WT) mice, as seen in previous studies of 3H9-Tg BALB/c mice (9). The 1 B cells in 3H9 knockin mice were also positive for IgMa, the IgM allotype of the transgenic IgH locus, suggesting that the 1 B cell population is likely to be autoreactive (Fig. 1B). 1 B cells comprise 
8% of the B220pos lymphocytes in 3H9 spleens versus 5% in WT controls (P < 0.001). Deficiency for FcRIIb in 3H9 animals further increases the splenic 1 B cell compartment up to 10–12% (P < 0.03), while non-transgenic FcRIIb–/– spleens have 1 B cell frequencies similar to WT controls (Fig. 1C). The finding that 3H9FcRIIb–/– mice have an increased frequency of 1 B cells compared with 3H9FcRIIb+/+ mice suggests that FcRIIb normally helps limit the expansion of autoreactive B cells.
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Further analysis of cell-surface markers expressed by splenic
1 B cells revealed decreased levels of IgM and IgD in all 3H9 mice, both with and without FcRIIb (Fig. 2). This surface phenotype is consistent with a history of previous antigen exposure (9). CD21 and CD23 surface levels on 1 B cells were also low in 3H9FcRIIb+/+ spleens, but not in 3H9FcRIIb-deficient animals (Fig. 2), and can be interpreted as the maturational arrest of an autoreactive B cell population in the presence of FcRIIb but not in its absence. The contrasting phenotype in 3H9FcRIIb–/– mice with intermediate levels of CD21 and slightly increased levels of CD23 is characteristic of mature follicular B cells (24). The phenotypic differences for CD21 and CD23 are readily apparent in aged animals but are harder to appreciate in younger groups of mice (data not shown), suggesting that the autoreactive follicular B cells accumulate over the lifetime of the FcRIIb–/– animals.
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Immunohistologic staining of splenic sections was performed to further characterize the
1 B cell population. In agreement with previous studies that discovered follicular exclusion of autoreactive B cell populations in BALB/c mice (9), 3H9FcRIIb+/+ B6 mice also had 1 B cells arrested at the T–B interface of splenic follicles (Fig. 3A). More than 80% of splenic follicles in both 3- and 9-month old 3H9FcRIIB+/+ spleens had unambiguous follicular exclusion of 1 B cells, an observation that correlates with the functional inactivation of potentially pathogenic cells (Table 1). In contrast, the 1 B cells in spleens of 3H9FcRIIb–/– mice were not efficiently arrested at the T–B interface (Fig. 3B), particularly in older animals. Even though 3H9FcRIIB–/– mice under 2 months of age had exclusion comparable to age-matched 3H9FcRIIB+/+ counterparts, 9-month-old animals lost follicular exclusion of 1 B cells in over 90% of splenic follicles (Table 1). These findings indicate that FcRIIb participates in the follicular exclusion of autoreactive B cells in an age-dependent manner.
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PNA staining of splenic sections detected substantially more germinal center activity in 9-month-old 3H9Fc
RIIB–/– mice than in 3H9FcRIIB+/+ controls (Fig. 3G and H). In addition, three-color immunostaining of 3H9FcRIIB–/– splenic tissue revealed 1 B cells within the germinal centers of the autoimmune mice (Fig. 3I). The observation that non-excluded B cells participate in germinal center reactions further suggests that regulation of peripheral autoreactivity is impaired in the absence of FcRIIB.
Anti-DNA antibody production
FACS analysis of splenic B cells revealed a distinct B220loCD138hi cell population in 9-month-old 3H9FcRIIb–/– mice that is characteristic of antibody-forming cells (AFCs) (Fig. 4A). This plasma cell population was much smaller in younger 3H9FcRIIb–/– females and in 3H9FcRIIb+/+ controls (Fig. 4B). To examine whether these apparent AFCs are capable of secreting anti-DNA antibodies, DNA enzyme-linked immunosorbent spot (ELISPOT) assays were performed on splenocytes of 9-month-old 3H9 mice sufficient or deficient for FcRIIb (Fig. 5). Although IgM anti-DNA AFC frequencies were not significantly different in either group, the number of anti-DNA-secreting splenic B cells of the IgG isotype was 8-fold higher in 3H9FcRIIb–/– mice compared with 3H9FcRIIb+/+ animals (P < 0.001). In concert with histologic loss of follicular exclusion in FcRIIb–/– spleens, the age-dependent increase in autoreactive AFCs implicates a progressive failure of peripheral tolerance in 3H9FcRIIb–/– animals. DNA ELISPOT assays of BM cells indicated that only 3H9FcRIIb–/– animals had anti-DNA IgG and IgM AFCs in their marrow, and more so in older animals (ranging from 5 to 55 spots per million BM cells). In contrast, no increase of anti-DNA AFCs were detected in 3H9FcRIIb+/+ marrow over WT (0–3 spots per million). These findings suggest that the splenic AFCs in 3H9FcRIIb+/+ mice may be short-lived plasma cells, whereas 3H9FcRIIb–/– mice have autoreactive AFCs in both spleen and marrow, likely constituting a mixture of both long- and short-lived plasmacytes and thus suggestive of a maturing autoimmune response.
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Not only do 3H9Fc
RIIb–/– mice have the potential to produce auto-antibodies, as suggested by their increased frequency of splenic AFCs measured in DNA ELISPOT, but these cells may be active in vivo, as suggested by DNA ELISA experiments (Fig. 6). ELISA data indicate that sera from 9-month-old 3H9FcRIIb–/– mice have significantly higher IgG and IgM anti-DNA levels compared with 3H9FcRIIb+/+ controls (P = 0.04 and P = 0.01, respectively). Once again, this autoreactivity evolves over time; there is no significant differences in serum auto-antibody levels in the younger animals. The presence of IgG anti-DNA antibodies circulating in aged 3H9FcRIIb–/– mice and the ability of 3H9FcRIIb–/– splenocytes to secrete IgG auto-antibodies in ELISPOT assays imply that the non-excluded 1 B cells in 3H9FcRIIb–/– mice are able to undergo class switching and terminal differentiation into autoreative plasmacytes.
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Role of myeloid versus lymphoid expression of Fc
RIIb in regulation of autoreactive B cellsSince Fc
RIIb is expressed on a wide variety of cell types including B cells and myeloid cells, it is of interest to determine whether loss of B cell tolerance in 3H9FcRIIb–/– mice is B cell intrinsic. Two cellular transfer models were implemented to help distinguish between B cell- and myeloid cell-dependent effects. In the first model, BM from either 3H9FcRIIb+/+ or 3H9FcRIIb–/– mice was transferred into lethally irradiated WT recipients. Histologic analysis of recipient spleens 9 months after transfer showed that only FcRIIB+/+ 1 B cells arrest at the T–B interface of splenic follicles (Fig. 3C). In contrast, B6 mice that received marrow from 3H9FcRIIb–/– animals lost follicular exclusion of autoreactive FcRIIb–/– 1 B cells (Fig. 3D), similar in appearance to the spleens of the respective non-chimeric donor strains (Fig. 3A and B and Table 1).
ELISPOT analysis of splenocytes of chimeric mice 9 months after transfer of BM from either 3H9FcRIIb+/+ or 3H9FcRIIb–/– mice was also comparable to the data from the non-chimeric animals. Splenic IgM anti-DNA AFCs were detected by ELISPOT assay in both cases, regardless of the presence or absence of FcRIIb on the BM donor cells. However, those recipients that received 3H9FcRIIb–/– marrow had higher numbers of IgG anti-DNA AFCs than those that received 3H9FcRIIb+/+ marrow (173 versus 63; P = 0.07; Table 2). These transfer experiments suggest that follicular exclusion requires FcRIIb on donor BM cells, but they do not discriminate whether the B cells themselves or other donor-derived cell types are responsible for the effect.
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A second transplant approach was developed to investigate the relative contributions of B cell versus myeloid cell Fc
RIIb status. In this mixed chimera model, BM of either 3H9FcRIIb+/+ or 3H9FcRIIb–/– mice was mixed with cells of RAG1–/– marrow in a ratio of 1:10 before intravenous transfer into lethally irradiated B6 recipients. Under these conditions, the B cell compartment in the chimeric animal is derived exclusively from the 3H9 donor, and is either FcRIIb+/+ or FcRIIb–/–, accordingly. In contrast, the myeloid compartment is a mixed population of which 90% initially originated from FcRIIb+/+ RAG1–/– marrow. This mixed chimeric design separates FcRIIb expression on myeloid cells (FcRIIb+/+) from lymphoid cells (FcRIIb–/– or FcRIIb+/+) and allows one to ask whether follicular exclusion is a B cell-intrinsic phenomenon.
Splenic histology was analyzed 9 months after the mixed BM chimeras were made. In mice repopulated with 3H9FcRIIb+/+ marrow, the 1 B cells were clearly excluded from 60% of splenic follicles. In contrast, the experimental group that received 3H9FcRIIb–/– B cells with FcRIIb+/+ myeloid cells retained 1 B cell exclusion in only 12% of follicles (Fig. 3E and F and Table 1). These mixed BM chimeras demonstrate that FcRIIb on the lymphoid lineage—most likely on B cells—is needed for follicular exclusion of autoreactive 1 B cells from the splenic follicle and that FcRIIb–/– 1 B cells escape follicular exclusion.
Unlike the histology data suggesting that follicular exclusion of autoreactive B cells requires B cell expression of FcRIIb, ELISPOT examination of the mixed chimeric spleens produced equivocal results. Both IgM and IgG anti-DNA AFCs were detected by ELISPOT analysis 9 months after transfer, regardless of the presence or absence of FcRIIb on BM-derived cells (Table 2). However, none of the chimeras had elevated IgG auto-antibody titers, possibly suggesting that the early implementation of peripheral tolerance (represented by follicular exclusion) is FcRIIb dependent and that additional FcRIIb-independent checkpoints further regulate class switching and terminal differentiation into autoreactive plasma cells.
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Discussion |
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Autoreactive B cells in WT mice are controlled by several tolerogenic mechanisms including deletion, anergy and BCR editing. In cases where lower affinity autoreactive B cells escape such central checkpoints or arise de novo via somatic hypermutation, peripheral strategies are needed to pre-empt the emergence of pathogenic autoreactivity. Peripheral tolerance has been studied extensively in the 3H9 transgenic mouse model where the 3H9-IgM transgene was randomly inserted in the genome of BALB/c mice (4). The data presented here expand those observations by demonstrating a role for Fc
RIIb in the maintenance of B cell tolerance. The original 3H9 animal model was adapted in two ways for the current experiments. First, the 3H9 VDJ knockin mouse (23) was used instead of the original 3H9-IgM transgenic animal (4). This approach helps rule out the possibility that follicular exclusion is a transgene-dependent artifact arising from a randomly inserted Ig transgene construct that is incapable of normal class switching and somatic hypermutation. Second, since the autoreactive predisposition of FcRIIb deficiency manifests in B6 but not BALB/c animals (22), 3H9 transgenic animals were back-crossed 10 generations onto the susceptible genetic background.
Before embarking on this study, it was necessary to verify that 3H9FcRIIb+/+ B6 animals indeed retain peripheral regulation of autoreactive 1 B cells and maintain self-tolerance. This concern arose from parallel back-crossing experiments in which 56R, a 3H9-related VDJ transgene (25), is dysregulated when back-crossed from BALB/c onto B6 (26–28). In contrast to the 56R model system, the current data from 3H9FcRIIb+/+ B6 animals bear important similarities to the original 3H9 transgenic BALB/c model (9). Transgenic mice of both varieties have increased frequencies of autoreactive 1 B cells compared with WT controls and yet the animals are not autoimmune. The splenic anti-DNA B cells are regulated in part by follicular exclusion and have surface phenotypes consistent with maturational arrest (CD21loCD23lo) in the face of previous antigen exposure (IgMloIgDlo). The cell-surface phenotype and arrest at the T–B border are evidence of peripheral regulation; terminal differentiation of autoreactive B cells into antibody-secreting plasma cells is interrupted. Not only do 3H9 mice lack circulating IgG anti-DNA antibodies, but they also lack IgG anti-DNA-secreting cells in their spleens. These results corroborate serologic data from other investigators who have independently crossed 3H9 to the B6 background and also found tolerance preserved (27, 29). Together, these data support the proposal that follicular exclusion is a peripheral tolerogenic mechanism that helps prevent anti-DNA B cell class switching in germinal centers and thus helps protect the animals from progressive autoimmune disease.
Since the tolerant phenotype is essentially the same in the transgenic and the knock-in models, we were able to use the B6 3H9 mice to further explore the peripheral mechanisms important for regulating autoreactive splenic B cells. We chose to explore the role of FcRIIb in these mice because B6 mice—but not BALB/c animals—require this inhibitory ITIM receptor to remain self-tolerant (22). FcRIIb–/– B6 mice have severe autoimmune disease with high levels of anti-nuclear antibodies and progressive immune complex glomerulonephritis. By breeding B6 3H9 animals with FcRIIb–/– mice, one can ask whether FcRIIb is required for those peripheral tolerogenic mechanisms that protect 3H9 mice from developing autoimmune disease.
Unlike their FcRIIb+/+ counterparts, the 3H9FcRIIb–/– mice break tolerance and develop features of autoimmune disease. This is a progressive phenomenon that is not readily detected in young animals but is unequivocal by 9 months of age. While spleens from young 3H9FcRIIb–/– animals retain the ability to exclude most of their autoreactive 1 B cells from splenic B cell zones, older mice are overwhelmed such that follicular exclusion is lost. These non-excluded 1 B cells have the CD21 and CD23 surface phenotype consistent with mature follicular B cells (24). Similarly, in contrast to younger mice who better resemble the FcRIIb intact controls, older 3H9FcRIIb–/– animals have increased frequencies of plasma cells and auto-antibody-secreting cells as well as elevated titers of circulating anti-DNA auto-antibody. Moreover, since some anti-DNA antibodies in 3H9FcRIIb–/– females are of the IgG isotype, it is likely that the autoreactive B cells have participated in germinal center reactions. This inference is supported by the histologic detection of 1 immunoreactivity within PNA-positive regions of 3H9FcRIIb–/– spleens.
FcRIIb is present on cell types of both myeloid and lymphoid lineages. Previous studies have demonstrated that the lupus-like disease of FcRIIb–/– mice can be transferred with BM progenitor cells and have also shown that B cell autoreactivity emerges even in the context of FcRIIb intact myeloid cells (22). To further characterize the role of FcRIIb function in peripheral tolerance, the current study compares the effect of autoreactive B cells with and without FcRIIb on the fate of follicular exclusion. As predicted, BM transplant experiments in the 3H9 model system demonstrated that follicular exclusion transfers with 3H9FcRIIb+/+ but not with 3H9FcRIIb–/– progenitor cells. Results from the mixed chimera experiments transferring RAG1–/– cells together with 3H9 progenitors further suggest that follicular exclusion is B cell dependent. Exclusion occurs only when the 3H9 B cells are FcRIIb intact and is lost even when 3H9FcRIIb–/– B cells co-exist with FcRIIb+/+ myeloid cells. In concordance with this latter observation, recipients of FcRIIb–/– B cells also had mildly increased frequencies of splenic anti-DNA AFCs, but in contrast to published data (22), no significant IgG anti-DNA titers were detected. It must be formally acknowledged that neither these nor previous approaches rule out the possibility that small populations of donor-derived FcRIIb–/– myeloid cells surviving in the chimeric animals contribute to their autoimmune outcomes. It is even possible that discrepancies between experimental results relate in part to the relative ‘potencies’ of these residual populations. Nonetheless, experiments using retroviral-based reconstitution of FcRIIb expression in FcRIIb–/– mice support the interpretation that FcRIIb effects on tolerance are mediated in a B cell intrinsic fashion (30).
The data reported here also expand previous observations in the 56R anti-DNA transgenic mouse model mentioned above that implicated a role for FcRIIb in peripheral B cell tolerance (25, 27). The spontaneous albeit non-pathogenic anti-DNA autoreactivity of the 56R mice (26–28) is significantly exacerbated in the absence of FcRIIb to the point where 56RFcRIIb–/– animals develop lupus-like disease with immune complex glomerulonephritis (27). Data from those studies suggested a model where FcRIIb's role in peripheral tolerance depends on its ability to inhibit the secretion of IgG auto-antibody by autoreactive B cells. The current data from 3H9FcRIIb–/– mice suggest that the putative FcRIIb-dependent checkpoint may be more complex than initially envisioned, operating not only at the level of IgG auto-antibody secretion but also at an earlier stage of B cell activation. The fact that the presence or absence of follicular exclusion correlates with the presence or absence of FcRIIb implies that the receptor mediates inhibitory functions even before activated B cells enter splenic B cell areas and engage in germinal center reactions. In addition, the chromosomal environment of the disrupted FcRIIb gene is potentially rich in pro-autoimmune, 129-derived alleles of the nearby Sle1 locus on chromosome 1 (18, 31), some of which function in a B cell autonomous manner (32). It remains to be determined whether these linked genes modulate the FcRIIb-dependent phenotype.
The observations that follicular exclusion and other indicators of B cell tolerance are lost over time in 3H9FcRIIb–/– mice suggest that additional factors must modify FcRIIb's effect on B cell regulation. FcRIIb down-regulation has been reported in aging mice (33) and, more importantly, age-related changes in follicular exclusion were reported in 3H9-Tg BALB/c mice (34). From at least 11 to 24 months of age, these animals progressively lost follicular exclusion and produced auto-antibodies. Furthermore, age-dependent changes in splenic microarchitecture were noted even in non-transgenic aging animals, more so in WT B6 than BALB/c mice (34). Extrapolating from those observations, one might speculate that FcRIIb normally helps mitigate an inevitable age-related failure of B cell tolerance and in its absence, 3H9FcRIIb–/– spleens disorganize more rapidly.
In summary, these experiments demonstrate that FcRIIb participates in the peripheral regulation of autoreactive B cells. FcRIIb deficiency in 3H9 B6 mice leads to an accumulation of autoreactive splenic B cells that are inappropriately regulated compared with FcRIIB+/+ controls. Follicular exclusion of 1 B cells is impaired, the frequency of splenic anti-DNA AFCs is increased and IgG anti-DNA titers are elevated in 3H9FcRIIb–/– mice. The follicular exclusion of autoreactive 1 B cells seems to require FcRIIb expression on the B cells themselves. This tolerogenic role of FcRIIb may be one of the peripheral regulatory mechanisms that prevents autoreactive 1 B cells from entering germinal center reactions and thereby attenuates auto-antibody production.
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Acknowledgements |
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We thank M. Alimzhanov and R. Barrington for critical discussion and R. Yeamans for great animal care. This work was supported by the National Institutes of Health grant P01AI52343 and by a fellowship of the Deutsche Forschungsgemeinschaft NE895/1-1 to A.N.
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Abbreviations |
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| AFC, antibody-forming cell |
| B6, C57BL/6 |
| BCR, B cell receptor |
| BCIP, 5-bromo-4-chloro-3-indolylphosphate |
| BM, bone marrow |
| ELISPOT, enzyme-linked immunosorbent spot |
| FcRIIb, Fc receptor IIb |
| ITAM, immunoreceptor tyrosine-based inhibitory motif |
| PNA, peanut agglutinin |
| SLE, systemic lupus erythematosus |
| TE, Tris–EDTA |
| WT, wild type |
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Notes |
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* These authors contributed equally to this work.
Transmitting editor: R. S. Geha
Received 12 August 2006, accepted 9 January 2007.
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References |
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