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The Journal of Immunology, 2002, 168: 13-21.
Copyright © 2002 by The American Association of Immunologists

Low-Affinity Anti-Smith Antigen B Cells Are Regulated by Anergy as Opposed to Developmental Arrest or Differentiation to B-11

Michelle Borrero and Stephen H. Clarke2

Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC 27599


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Understanding the regulation of B lymphocytes specific for self-Ags targeted in human and murine systemic lupus erythematosus, such as the ribonucleoprotein Smith Ag (Sm), is crucial to understanding the etiology of this autoimmune disease. To address the role of B cell receptor affinity in the regulation of anti-Sm B cells, we generated low-affinity anti-Sm transgenic mice by combining the anti-Sm 2-12H transgene with a V{kappa}8 transgene. In contrast to 2-12H transgenic mice, in which anti-Sm B cells are predominantly splenic transitional, and peritoneal B-1, low-affinity anti-Sm B cells are long-lived B-2 cells and are found in the spleen, lymph nodes, and peritoneum. However, they are unresponsive to LPS in vitro, indicating that they are anergic, although they do not down-regulate IgM and are not excluded from follicles even in the presence of nonautoreactive B cells. Thus, low-affinity anti-Sm B cells appear to have a partial form of anergy. Interestingly, these cells have elevated levels of MHC class II and CD95, but not CD40, CD80, or CD86, suggesting that they are poised to undergo deletion rather than activation upon T cell encounter. These data identify anergy as a mechanism involved in anti-Sm B cell regulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Multiple mechanisms have evolved to ensure the regulation of autoreactive B cells. These include receptor editing, central and peripheral deletion, follicular exclusion, and anergy (1, 2, 3, 4, 5, 6, 7, 8, 9). What controls the mechanism used for a given autoreactive B cell is uncertain. Factors known to influence this choice include the concentration and form of the autoantigen, the affinity of the B cell receptor (BCR)3 for the self-Ag, the developmental stage of the B cell at the time of Ag encounter, the availability of T cell help, and the composition of the B cell repertoire (1, 10, 11, 12). Thus, identifying the mechanisms that regulate autoreactive B cells is crucial to understanding how tolerance is broken in autoimmunity.

Anergy is of particular interest because this mechanism retains autoreactive B cells in the repertoire, offering a large window of opportunity for the loss of tolerance. Not all anergic B cells are alike. For example, at one extreme are anergic anti-hen egg lysozyme (HEL) B cells. These B cells have down-regulated surface IgM and have a short half-life due to exclusion from B cell follicles (1, 4). In addition, the BCR is uncoupled from the signal transduction pathways in these cells (13). This contrasts with anergic anti-dsDNA B cells. Although similar to anti-HEL B cells in that they down-regulate surface IgM and are excluded from B cell follicles, they differ in that they developmentally arrest at the transitional B cell stage (7, 14). At the other extreme are anti-ssDNA B cells. They are long-lived B-2 cells that do not down-regulate surface IgM and can occupy splenic follicles (7, 15). Moreover, they retain at least some signal transduction potential (7, 15). Notwithstanding these differences, all anergic B cells share an impaired ability to differentiate to Ab-secreting cells (ASC) in response to Ag and LPS. Relevant to the development of autoimmunity is the fact that anergy is reversible and, thus, under the appropriate circumstances, anergic B cells could provide a source of autoreactive B cells that contribute to autoimmune disease (7, 16).

We have characterized the anti-Smith Ag (Sm) response in autoimmune MRL/lpr mice because it occurs in human and murine lupus (17, 18, 19). These mice spontaneously develop a systemic lupus erythematosus-like disease beginning at ~5 mo of age. These mice develop anti-Sm responses at a prevalence of ~25%, similar to that in human systemic lupus erythematosus (20, 21). To understand how anti-Sm B cells are regulated, we generated Ig H chain transgenic (Tg) mice (2-12H) (22). This H chain rearrangement derives from the anti-Sm hybridoma 2-12 of MRL/lpr origin (17, 18). Abs using 2-12H chains can bind Sm, ssDNA, both, or neither, depending on the L chain with which it is paired. Moreover, the L chain determines the affinity for these autoantigens, which ranges over 2–3 orders of magnitude (23).

2-12H Tg mice have large numbers of anti-Sm and anti-ssDNA B cells. In the spleen most of these cells are transitional, suggesting a block of differentiation to the mature B-2 cell stage (22). Interestingly, anti-Sm B cells also constitute ~30% of the peritoneal B-1 cell subset in these mice (24), yet the serum levels of anti-Sm Abs are low and not different from those of non-Tg mice. However, these cells appear to be functional, because anti-Sm Abs are secreted upon immunization with murine small nuclear ribonucleoproteins, and both transitional and B-1 anti-Sm B cells can be activated by LPS (22, 24). Hence, the absence of elevated levels of serum anti-Sm is a paradox, particularly due to the large number of peritoneal anti-Sm B-1 cells present in these mice. Differentiation to B-1 may be involved in the maintenance of anti-Sm B cell tolerance, possibly through the migration of anti-Sm B cells to the peritoneum and away from self-Ag or through the expression of CD5, a negative regulator of BCR signaling (24). Thus, anti-Sm B cells in 2-12H Tg mice appear to be regulated by developmental arrest and differentiation to B-1. While 2-12H Tg mice have been instrumental in the characterization of anti-Sm B cell regulation in nonautoimmune mice, the diversity of affinities that are conferred by the use of multiple endogenous L chains does not allow us to exclude the possibility that other mechanisms regulate cells of this specificity.

To elucidate the role of BCR affinity in anti-Sm B cell regulation, we interbred 2-12H and V{kappa}8 Tg mice. Abs composed of 2-12H and V{kappa}8 L chains have a low affinity for Sm and do not bind ss- or dsDNA (23). In contrast to anti-Sm B cells of 2-12H Tg mice, low-affinity anti-Sm B cells become mature long-lived B-2 cells and do not differentiate to B-1. They occupy B cell follicles in the spleen even when nonautoreactive B cells are a majority of the B cell repertoire. These cells are not spontaneously activated, as indicated by the low levels of transgene-encoded Ab in the serum, and, most significantly, they do not differentiate to ASC upon in vitro stimulation. Therefore, we conclude that they are anergic. Furthermore, they have up-regulated MHC class II and CD95 expression, suggesting that they are poised to undergo deletion, rather than activation, upon T cell encounter.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

2-12H Tg mice were created as previously described (22). V{kappa}8 Tg mice (25) were provided by Dr. M. Weigert (Princeton University, Princeton, NJ). Both 2-12H and V{kappa}8 Tg mice were bred with C{kappa}-/- mice (26) (provided by GenPharm International, San Jose, CA) to limit the endogenous light chain use to {lambda}. 2-12H C{kappa}-/- mice were then bred to V{kappa}8 C{kappa}-/- to create 2-12H/V{kappa}8 double-Tg mice (V{kappa}8 Dbl Tg). Experiments were performed on F1 mice of this cross. All mice were 2–5 mo of age at the time of analysis. Animals were housed and bred in a conventional facility at University of North Carolina (Chapel Hill, NC).

Screening of 2-12 and V{kappa}8 transgenes was performed by PCR as previously described (22, 25). C{kappa}-/- mice were detected by PCR using primers that amplified the J{kappa}2 and J{kappa}3 regions of the wild-type {kappa} locus. A 649-bp product was produced in mice that had one or more copies of the wild-type {kappa} locus. Primers sequences are: J{kappa} forward, 5'-CGGTGCTCAGACCATGCTCAG-3'; and J{kappa} reverse, 5'-GCCTGAGGCTGATCCAGTTAGAT-3'. The PCR included 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 1 µM of each primer, and 2.5 U Taq DNA Polymerase (Life Technologies, Gaithersburg, MD). Product amplification was for 5 min at 94°C, 30 s at 94°C, 1 min at 62°C, and 1 min at 70°C. A total of 30 cycles of amplification were performed. The final extension was for 7 min at 70°C.

Flow cytometric analysis

Spleens and lymph nodes were crushed between the frosted ends of two slides to make single-cell suspensions. Bone marrow cells were extracted from the femur and tibia of the two hind legs, and peritoneal cells were isolated by lavage. After cell suspensions were made in HBSS, RBCs were lysed using a solution that contained 0.15 M ammonium chloride, 1.0 mM potassium bicarbonate, and 0.1 mM EDTA. FcR were blocked using mAb 2.4G2. Cells were incubated for at least 10 min at 4°C. Staining was performed in HBSS supplemented with 3% FBS (Life Technologies).

The Abs used for flow cytometry were against IgMa (DS-1), IgMb (AF6-78), {kappa} (R5-240), CD5 (53-7.3), CD45R/B220 (RA3-6B2), CD23 (B3B4), CD24 (M1/69), CD43 (S7), CD21/35 (7G6), CD40, CD95, CD80, CD86, CD22, CD44, and IAb/d and were obtained from BD PharMingen (San Diego, CA). These Abs were biotinylated or conjugated to allophycocyanin, PE, FITC, or CyChrome. For the identification of anti-Sm B cells we used Sm (SMA-3000; Immunovision, Springdale, AR) that was biotinylated in our laboratory as described previously (22). For the detection of biotinylated probes we used streptavidin-FITC (Jackson ImmunoResearch Laboratories, West Grove, PA), streptavidin-PerCP (BD PharMingen), or streptavidin-Cy5 (Calbiochem, La Jolla, CA). Three- and four-color analyses were performed at the University of North Carolina Flow Cytometry Facility (Chapel Hill, NC) using a FACScan and a FACSCalibur (BD Biosciences, Mountain View, CA), respectively. Analysis software was purchased from Cytomation (Fort Collins, CO).

BrdU labeling

Mice were given 0.5 mg/ml 5-bromo-2'-deoxyuridine (BrdU; Sigma-Aldrich, St. Louis, MO) with 1 mg/ml dextrose in their drinking water continuously for 8 wk. At different time points mice were sacrificed and spleens were isolated for analysis. Cell preparations and surface staining of 1 x 107 cells was done as described above. Staining for BrdU was done as described by Allman et al. (27) using anti-BrdU-FITC (BD Biosciences, San Jose, CA).

ELISA

Anti-Sm Abs in serum or tissue culture supernatants were measured using the protocol described by Eisenberg et al. (28). Briefly, 96-well flat-bottom polyvinylchloride plates were coated with Sm protein (Immunovision) for at least 5 h in borate-buffered saline. All washes were performed with borate-buffered saline. Plates were blocked with borate-buffered saline, 0.5% BSA, and 0.4% Tween 80 for a minimum of 1 h before adding the samples. All samples were analyzed in duplicate. To develop the assays we used either goat anti-mouse IgM-alkaline phosphatase (AP; Southern Biotechnology Associates, Birmingham, AL) or anti-IgMa-biotin followed by streptavidin-AP (BD PharMingen). Paranitrophenyl phosphate (1 mg/ml; Sigma-Aldrich) in 0.01 M diethanolamine was used to develop the assays. OD readings were taken at 405/600 nm.

For the detection of 2-12H/V{kappa}8-encoded Ab, plates were coated with rat anti-mouse {kappa} (BD PharMingen), and detection was made using biotinylated rat F(ab')2 anti-mouse IgMa (HB100, Bet-1; American Type Culture Collection, Manassas, VA) with streptavidin-AP. Total IgM was detected by coating with goat anti-mouse IgM and developing with the same reagent coupled to AP (Southern Biotechnology Associates). To measure total IgMa or IgMb, plates were coated with purified anti-IgMa or anti-IgMb (BD PharMingen) and developed with anti-IgM-AP. Purified mouse IgMa (TEPC 183; Sigma-Aldrich) was used as a standard for total IgM and IgMa ELISAs. The IgMb standard (CBPC112) was a gift from Dr. P. Cohen (University of Pennsylvania, Philadelphia, PA).

LPS stimulation

Spleen cells were harvested, and RBC were lysed using RBC lysis buffer described in the flow cytometry section. Cells were resuspended in DMEM-high glucose at 1 x 106 cells/ml. Triplicate cultures were set for 72 h in the presence or the absence of 50 µg/ml LPS. Supernatants were analyzed by ELISA.

Immunofluorescence

Freshly isolated spleens were imbedded in Tissue-Tek OCT (Sakura Finetek, Torrance, CA) and frozen in 2-methyl-butane and liquid nitrogen. Five-micrometer spleen sections were prepared and acetone-fixed before staining. Sections were blocked for 30 min with 6% BSA in PBS. Staining was performed for 1 h at room temperature with anti-CD3-FITC, anti-B220-allophycocyanin, and anti-IgMa or IgMb-biotin (BD PharMingen). After the slides were rinsed with PBS, the sections were stained with streptavidin-Cy3 (Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Fluorescein signal was enhanced using the Alexa Fluor 488 signal amplification kit for FITC-conjugated probes (Molecular Probes, Eugene, OR) as described by the manufacturer, and the slides were then mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA). Analysis was performed at the Physiology Imaging and Neuroscience Confocal Facility at University of North Carolina, using a Leica TCS-NT laser scanning confocal microscope (Leica, Deerfield, IL).

Bone marrow chimeras

Hematopoietic stem cells from 2- to 3-mo-old V{kappa}8 Dbl Tg mice and B6 non-Tg mice were isolated as described by Goodell et al. (29). Briefly, bone marrow cells were extracted, and RBCs were lysed as described for flow cytometric analysis. Cells were resuspended at 1 x 106 cells/ml in RPMI 1640 medium (HyClone Laboratories, Logan, UT) with 2% FBS and 5 µg/ml Hoechst 33342. Staining was performed at 37°C for 90 min. Propidium iodide at 2 µg/ml was added just before analysis. Analysis and sorting of side population cells, which exclude the Hoechst dye and are enriched for hemopoietic stem cells, were performed with a MoFLo high speed sorter (Cytomation). Cells (2 x 103) in PBS were injected via tail vein into B6 RAG-1-/- mice (The Jackson Laboratory, Bar Harbor, ME) that had been given 600 rad 24 h previously. Analysis of recipients was performed at least 8 wk after reconstitution.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Low-affinity anti-Sm B cells are long-lived mature B-2 cells

We have previously demonstrated that 2-12H chains paired with V{kappa}8L chains bind Sm with low affinity (23). Thus, to generate low-affinity anti-Sm Tg mice, 2-12H and V{kappa}8 Tg mice were bred to generate double-Tg mice (V{kappa}8 Dbl Tg). The L chain encoded by the V{kappa}8 transgene is identical with the V{kappa}8 L chain used in the earlier in vitro analysis (23, 25). Thus, the anti-Sm Abs produced by V{kappa}8 Dbl Tg mice will be low affinity. These mice were also bred to C{kappa}-/- to ensure that the all {kappa} L chains are of transgene origin. Unless otherwise indicated, all V{kappa}8 Dbl and V{kappa}8 Tg mice used in this study are {kappa}-/-, and all control 2-12H Tg and non-Tg mice are {kappa}+/+. As shown in Table IGo, total spleen cell numbers in V{kappa}8 Dbl Tg mice were not significantly different from those in 2-12H, V{kappa}8 Tg, and non-Tg mice, although, as is typical of Ig Tg mice, the total number of B cells were significantly lower than those in non-Tg mice (p < 0.005).


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  		Table I. Total splenic B cells in V{kappa}8 Dbl Tg mice1

 
To assess the expression of the H and L chain transgenes by B cells in V{kappa}8 Dbl Tg mice, spleen cells were stained with Abs specific for IgHa (the 2-12H allotype) and {kappa}. As shown in Fig. 1GoA, >93% of splenic B220+ cells of V{kappa}8 Dbl Tg mice express IgMa{kappa}. The remaining B cells express b allotype H chains or {lambda}-chains (data not shown). Nearly all (>95%) lymph node B cells are also IgMa{kappa} (data not shown). Thus, by virtue of expression of both transgenes the majority of splenic and lymph node B cells in V{kappa}8 Dbl Tg mice are anti-Sm. The ability of these B cells to bind Sm was directly assessed by flow cytometry using biotinylated Sm. As shown in Fig. 1GoB, only the IgMhigh B cells exhibit Sm staining, despite the fact that nearly all splenic B cells of V{kappa}8 Dbl Tg mice are anti-Sm. Thus, the lack of Sm staining by the IgMlow B cells must be due to their low surface IgM level. As previously demonstrated, ~30% of 2-12H splenic B cells stain with biotinylated Sm (22) (Fig. 1GoB). Anti-IgHa-specific Abs are used to follow the development and regulation of these low-affinity anti-Sm B cells, because essentially all IgHa B cells express the V{kappa}8 transgene and thus are low-affinity anti-Sm B cells.



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  		FIGURE 1. Flow cytometric analysis of V{kappa}8 Dbl Tg splenic B cells. A, Transgene use was assessed by staining with Abs specific for the H chain allotype of the a allotype (IgMa) and mouse {kappa}. B, The ability of V{kappa}8 Dbl Tg B cells to bind Sm was determined using biotinylated Sm. Transgenic B cells are identified using anti-IgMa Ab. All histograms in A and B are gated on B220+ cells. C, The developmental stage of splenic B cells was determined using four-color flow cytometric analysis by assessing the expression of B220, IgMa or IgMb, CD23, and CD21/35. The bottom panels are gated on either CD23+ or CD23- populations. The differential expression of IgM and CD21/35 by CD23- B cells allows the identification of transitional 1 and MZ B cells (bottom left panel), and the differential expression of IgM and CD21/35 by CD23+ B cells allows the identification of transitional 2 and mature (M) B cells (bottom right panels) (30 ). Both V{kappa}8 Dbl Tg (left panels) and V{kappa}8 Tg mice (right panels) were analyzed.

 
The differentiative potential of low-affinity anti-Sm B cells was determined by examination of differentiation marker expression. These markers include CD23, heat-stable Ag; (HSA; CD24), CD21/35, CD5, and CD43, as well as IgM and B220. The majority of splenic B220+ cells (~79%) from V{kappa}8 Dbl Tg mice have a mature B-2 cell phenotype. They are CD23+, HSAlow, CD21/35low, CD5-, and CD43- (Fig. 1GoC and data not shown). In addition, some (~6%) are HSAhighCD21/35- transitional 1 cells, and a few (~1%) are HSAhighCD21/35low transitional 2 cells (30). Essentially no CD21/35highCD23- or CD5+CD43+ cells are detectable, indicating that marginal zone (MZ) B cells and B-1 cells contribute little or not at all to the splenic repertoire. In contrast, MZ (Fig. 1GoC) and B-1 cells (data not shown) are present in the spleens of V{kappa}8 Tg mice.

Anti-Sm B cells of 2-12H Tg mice differentiate to B-1 and constitute ~30% of the peritoneal B-1 repertoire (24). To determine whether low-affinity anti-Sm B cells differentiate to B-1, we examined peritoneal cells of V{kappa}8 Dbl Tg mice for evidence of differentiation to B-1. As shown in Fig. 2Go, low-affinity anti-Sm B cells in the peritoneum have a mature B-2 cell phenotype. They are CD23+IgMlowB220high and do not express the B-1 cell markers CD5 and CD43 (Fig. 2Go). Thus, in contrast to anti-Sm B cells in 2-12H Tg mice, low-affinity anti-Sm B cells do not differentiate to B-1.



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  		FIGURE 2. Low-affinity peritoneal anti-Sm B cells do not differentiate to B-1. Peritoneal B cells were analyzed by flow cytometry using Abs specific for B220, IgMa, IgMb, Sm, and CD5, CD23, and CD43. All histograms are gated on B220+ cells. The histograms in the second column are also gated on the IgMa (2-12H and V{kappa}8 Dbl Tg mice) or IgMb (V{kappa}8 Tg mice) cells. Histograms in columns 3–5 of 2-12H Tg mice are also gated on Sm-binding cells. Those of V{kappa}8 Dbl and V{kappa}8 Tg mice remain gated on the IgMa or IgMb cells. The percentage of cells in each box is given. Data for each were acquired from cells pooled from two or three Tg mice.

 
Because turnover rates of autoreactive B cells can be significantly shorter than those of nonautoreactive B cells (31), we determined the turnover rates of low-affinity anti-Sm B cells by BrdU incorporation. As shown in Fig. 3Go the turnover rate of transitional (HSAhigh) B cells of V{kappa}8 Dbl Tg mice suggests they have a half-life of ~3 days, significantly shorter (p < 0.0001) than the 2-wk half-life of their non-Tg C{kappa}-/- counterparts. The half-life of mature low-affinity anti-Sm B cells is estimated to be ~5 wk, not significantly different from the 7 wk estimated for B cells of non-Tg C{kappa}-/- littermates. Thus, peripheral deletion or developmental arrest do not appear to have a role in the regulation of low-affinity anti-Sm B cells, as they acquire a mature phenotype and are long-lived.



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  		FIGURE 3. Splenic low-affinity anti- Sm B cells have a long half-life. Incorporation of BrdU by transitional (HSAhigh; left) and mature (HSAlow; right) B cells was measured to determine the turnover rate. The half-life, which corresponds to the time at which 50% of the cells have incorporated BrdU, is indicated (dotted lines). Two to four V{kappa}8 Dbl Tg (thick line) and non-Tg C{kappa}-/- (thin line) mice were analyzed for each time point. Bars represent the SD.

 
Low-affinity anti-Sm B cells show evidence of Ag encounter but appear to be nonfunctional

To determine whether low-affinity anti-Sm B cells are activated in vivo, we examined the levels of transgene-encoded (IgMa{kappa}) Ab in circulation. Assaying for IgMa{kappa} Abs in the serum of V{kappa}8 Dbl Tg mice is more sensitive than determining the presence of anti-Sm Abs due to the low affinity of 2-12H/V{kappa}8 Abs for Sm. As shown in Fig. 4GoA, the total serum IgM Ab level in V{kappa}8 Dbl Tg mice, which includes both IgMa and endogenous IgMb Abs, is ~100-fold lower than that in non-Tg C{kappa}-/- littermates (p = 0.0019) and non-Tg BALB/c mice (p < 0.0001). It is ~3-fold lower than that in 2-12H Tg mice (p = 0.0057). More significantly, IgMa{kappa} Ab in V{kappa}8 Dbl Tg mice, which in these mice is anti-Sm, is ~10-fold lower than that in 2-12H Tg (p = 0.0005) and ~100-fold lower (p < 0.0001) than that in BALB/c mice despite the fact that the nearly all V{kappa}8 Dbl Tg B cells are IgMa{kappa} (Figs. 4GoB and 1B). Thus, V{kappa}8 Dbl Tg mice have very little anti-Sm Ab in the circulation, suggesting that either low-affinity anti-Sm B cells have not encountered Ag and are ignorant or that they have encountered Ag and are anergic.



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  		FIGURE 4. Analysis of IgM and transgene-encoded Abs in the sera of V{kappa}8 Dbl Tg mice. A, IgM (a and b allotypes) in the sera of V{kappa}8 Dbl Tg and control mice was quantified by ELISA. B, Serum samples were also tested for the presence of IgMa/{kappa} Abs. IgMa/{kappa} Abs represent low-affinity anti-Sm Abs (transgene encoded) in V{kappa}8 Dbl Tg mice. BALB/c mice represent non-Tg controls, because they endogenously express IgMa. Each circle represents an individual mouse. Bars represent the median of the samples tested. Statistical significance was assessed by Bonferroni pair comparisons. *, Statistically significant (p < 0.01) differences between V{kappa}8 Dbl Tg mice and the control groups.

 
To discriminate between anergy and ignorance, the expression levels of a variety of activation markers on V{kappa}8 Dbl Tg B cells were examined. As shown in Fig. 5Go, the expression levels of CD40, CD80 (B7-1), and CD86 (B7-2) on V{kappa}8 Dbl Tg B cells are slightly lower than or equal to those on V{kappa}8 Tg control B cells. In addition, the level of IgM is somewhat higher than it is on B cells of V{kappa}8 Tg mice, indicating that there is no down-regulation of IgM on these autoreactive B cells (Fig. 5Go). However, V{kappa}8 Dbl Tg B cells have higher levels of MHC class II and CD95 than V{kappa}8 Tg littermates. Because both MHC class II and CD95 expression levels can be elevated in response to Ag stimulation (32, 33), the elevated expression of these markers on low-affinity anti-Sm B cells suggests that they have encountered Ag in vivo.



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  		FIGURE 5. The expression of class II and CD95 (Fas) suggests Ag encounter by low-affinity anti-Sm B cells. V{kappa}8 Dbl Tg (solid line) and V{kappa}8 Tg (dotted line) splenocytes were analyzed by flow cytometry for expression of the activation markers CD40, CD86 (B7-2), CD80 (B7-1), class II (IAb/d), CD95 (Fas), and IgM. All histograms are gated on B220+ cells.

 
To directly determine the functional competence of low-affinity anti-Sm B cells, splenocytes of V{kappa}8 Dbl Tg mice were cultured with LPS for 3 days and Ab production was measured by ELISA. As shown in Fig. 6Go, low-affinity anti-Sm B cells from V{kappa}8 Dbl Tg mice do not secrete IgM upon LPS stimulation. This was assessed by an IgM-specific ELISA that detects both IgMa and IgMb Abs and by an anti-Sm ELISA. This result contrasts with the production of IgM by B cells of nonautoreactive control mice and with the production of anti-Sm Abs by B cells from 2-12H Tg mice (Fig. 6Go). Similar results were obtained with supernatants of 7-day LPS cultures (data not shown). Thus, low-affinity anti-Sm B cells of V{kappa}8 Dbl Tg mice are unresponsive to mitogen stimulation and therefore are anergic.



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  		FIGURE 6. Low-affinity anti-Sm B cells are unresponsive to LPS stimulation. Splenocytes from V{kappa}8 Dbl Tg mice were cultured with 50 µg/ml LPS for 3 days, and supernatants were analyzed by ELISA for the presence of IgM (left) and anti-Sm (right) Abs. Cultures were performed and analyzed in triplicate. A total of four to six mice in each control group and 10 V{kappa}8 Dbl Tg mice were analyzed. Bars represent the SEM.

 
Follicular exclusion is not involved in the regulation of low-affinity anti-Sm B cells

Exclusion from B cell follicles can contribute to the regulation of autoreactive B cells. Both anti-HEL B cells and anti-dsDNA B cells are excluded from B cell follicles (1, 14). To determine whether follicular exclusion plays a role in the regulation of anergic anti-Sm B cells in V{kappa}8 Dbl Tg mice, the localization of low-affinity anti-Sm B cells was determined. Spleen sections of V{kappa}8 Dbl Tg mice were stained with fluorescent Abs against CD3 and B220 to identify T cell and B cell areas, respectively. Transgenic IgMa B cells were identified using a biotin-labeled anti-IgMa Ab and streptavidin-Cy3 (red) for detection. As shown in Fig. 7GoA, IgMa+ B cells of V{kappa}8 Dbl Tg mice are located in follicles alongside T cell-rich periarteriolar lymphoid sheaths.



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  		FIGURE 7. Anergic anti-Sm B cells localize to B cell follicles. Frozen spleen sections were stained with Abs against CD3 (green), B220 (blue), and IgMa or IgMb (red) to identify the location of Tg+ B cells in the splenic white pulp. B cell follicles (F) and periarteriolar lymphoid sheaths (T) are noted. A, IgMa+ B cells localize to B cell follicles (pink cells) in V{kappa}8 Dbl Tg mice (right). A section of V{kappa}8 Tg spleen showing the location of IgMb+ cells is shown as a control (left). Images are at x10 magnification. B, Spleen sections of chimeric mice that were reconstituted with 25% V{kappa}8 Dbl Tg stem cells were stained as described above. IgMa+ cells stained red at x20 magnification are shown.

 
Follicular localization of autoreactive B cells can be influenced by the presence of nonautoreactive B cells (1, 34). Thus, the presence of low-affinity anti-Sm B cells in follicles could be due to a lack of nonautoreactive "competitor" B cells. To determine whether competition by nonautoreactive B cells alters the follicular localization of low-affinity anti-Sm B cells, bone marrow chimeric mice were generated. To create the chimeras, bone marrow stem cells from V{kappa}8 Dbl Tg and non-Tg mice were enriched and sorted as described by Goodell et al. (29), mixed at various ratios, and injected i.v. into sublethally irradiated B6 RAG-1-/- mice. Chimeric mice were analyzed 8 wk later. Immunofluorescence of spleen sections shows that low-affinity anti-Sm B cells identified as IgMaB220+ cells are intermixed among B220+IgMa- B cells in follicles (Fig. 7GoB). Thus, nonautoreactive B cells have no effect on the follicular localization of low-affinity anti-Sm B cells.

The presence of nonautoreactive B cells also does not alter the differentiation of low-affinity anti-Sm B cells. Analysis of IgMa and IgMb B cells in chimeric mice confirms that the mice are chimeric (Fig. 8GoA). The majority of low-affinity anti-Sm B cells (IgMa cells) have a mature B-2 cell phenotype (CD23+HSAlow) regardless of the ratio of Tg to non-Tg stem cells used for reconstitution (Fig. 8GoB). The same results were obtained with lymph nodes and peritoneal B cells (data not shown). Moreover, the anti-Sm B cells of chimeric mice do not contribute significantly to serum Ab levels; IgMb Abs are present in circulation even in mice that were reconstituted with mostly IgMa stem cells, but IgMa Abs are not detectable (limit of detection, 2.5 ng/ml) regardless of the stem cell ratio (Fig. 8GoC). Finally, IgMa B cells in chimeric mice do not respond to LPS (Fig. 8GoD), indicating that they remain anergic. Thus, the presence of nonautoreactive B cells does not alter the trafficking and regulation of low-affinity anti-Sm B cells.



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  		FIGURE 8. Competition by nonautoreactive B cells does not alter the phenotype or anergic state of V{kappa}8 Dbl Tg B cells. A, The percentage of total IgMa ({blacksquare}) and IgMb ({square}) B cells that were recovered from chimeric mice is shown. The percentage of V{kappa}8 Dbl Tg stem cells that were used for reconstitution is shown on the x-axis. Error bars represent the SEM. B, Three-color flow cytometric analysis of splenocytes from chimeric mice reconstituted with V{kappa}8 Dbl Tg and non-Tg stem cells at different ratios. Histograms are gated on IgMa+ (upper panels) or IgMb+ (lower panels) cells, which represent V{kappa}8 Dbl Tg or non-Tg-derived B cells, respectively. The percentage of stem cells refers to percentage of V{kappa}8 Dbl Tg stem cells that were injected into recipient mice (right panels, 25%; upper left panel, 100%; bottom left, 0%). The percentages of transitional 1 and 2 (T1 and T2), and mature (M) B cells are indicated. C, Serum of reconstituted mice was quantified by ELISA for the presence of both IgMa ({blacksquare}) and IgMb ({square}) Abs. D, Triplicate splenocyte cultures from chimeric mice were tested by ELISA for the presence of IgMa and IgMb Abs. Cells were cultured for 3 days in the presence and the absence of 50 µg/ml LPS. Error bars represent the SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present work we have characterized the regulation of low-affinity anti-Sm B cells. These B cells are long-lived, mature, follicular B-2 cells. They have elevated expression levels of MHC class II and CD95, suggesting that they have encountered Ag, but their inability to differentiate to ASC by LPS stimulation in vitro indicates that this Ag encounter has induced anergy. This is consistent with the extremely low serum IgM level in these mice. Significantly, the cell surface IgM expression on these B cells is slightly elevated relative to that in normal B cells, and the presence of nonautoreactive B cells does not alter the follicular localization, development, or anergic state of these cells. This is in sharp contrast to anergic anti-HEL B cells that have down-regulated surface IgM and are excluded from follicles by the presence of a majority of nonautoreactive B cells (1, 4). It is also in contrast to some anti-dsDNA that also down-regulate surface IgM and are excluded from follicles (35). The phenotype and behavior of anergic anti-Sm B cells more closely resemble those of anergic anti-ssDNA B cells (7, 15), a similarity made significant by the fact that the anergy of anti-ssDNA B cells is broken in murine lupus (36). Thus, based on the idea that anergy encompasses a spectrum of phenotypes (7, 37), we place anergic anti-Sm B cells along with anergic anti-ssDNA B cells at the lower end of the anergy spectrum.

The range of phenotypes that has been characterized for anergic B cells correlates with changes in BCR signaling capabilities. A weak tolerogenic signal appears to affect only some signaling capabilities, whereas a stronger signal appears to have a more significant effect on signaling. Anergic anti-HEL B cells, which receive a strong tolerogenic signal (38), show evidence of disruption in early signaling events, such as the partition of the BCR into lipid rafts (13). Anergic anti-ssDNA B cells of 3H9/V{kappa}8 Tg mice, which presumably receive a weak tolerogenic signal (7), flux Ca2+ and phosphorylate a number of intracellular proteins upon BCR cross-linking. Neither a Ca2+ flux nor protein phosphorylation is seen in anergic anti-dsDNA or anti-HEL B cells following IgM cross-linking (7, 38). These biochemical differences in BCR signaling capabilities may be significant to the etiology of lupus, because anergic B cells specific for lupus-associated Ags have biochemical changes resulting from weak tolerogenic signals that may be more easily overridden. In this regard the anergy of anti-ssDNA B cells is overcome in MRL/lpr mice (36). We are currently examining the signaling capability of these anergic anti-Sm B cells.

The induction of anergy in V{kappa}8 Dbl Tg mice contrasts with the regulation of anti-Sm B cells in 2-12H Tg mice. Most splenic anti-Sm B cells of 2-12H Tg mice appear to developmentally arrest at the transitional B cell stage (22). However, other anti-Sm B cells differentiate to B-1, constituting ~30% of the peritoneal B-1 population (24). There is no evidence of developmental arrest or differentiation to B-1 in V{kappa}8 Dbl Tg mice. Thus, anergy is a third pathway for the regulation of anti-Sm B cells. There are likely to be anergic anti-Sm B-2 cells in 2-12H Tg mice, because transgene-expressing B cells are free to use the available L chain repertoire. However, they are likely to be difficult to detect by flow cytometry, because, like V{kappa}8 Dbl Tg B cells, they probably do not stain well with biotinylated Sm. However, such cells may be detectable with biotinylated Sm as they pass through the IgMhigh transitional B cell stage. In this regard, we note that 2-12H Tg mice have an IgMhigh intermediate Sm-binding population (Fig. 1Go), which may consist of low-affinity anti-Sm B cells of the type described here.

The strength of the tolerogenic signal is likely to be the determining factor in how anti-Sm B cells are regulated. Because the availability of Ag in 2-12H and V{kappa}8 Dbl Tg mice is unlikely to be different, the affinity of the BCR for Sm must determine the mechanism of regulation. Both the transitional and B-1 anti-Sm B cells of 2-12H Tg mice appear to be of higher affinity than those of V{kappa}8 Dbl Tg mice, as the cells of both populations from 2-12H Tg mice stain brighter with biotinylated Sm than do those from V{kappa}8 Dbl Tg mice (Fig. 1GoB). This difference cannot be due to the level of surface IgM, because the transitional anti-Sm B cells of V{kappa}8 Dbl Tg mice have the same or a higher surface IgM level as many transitional anti-Sm B cells of 2-12H Tg mice (Fig. 1Go, A and B). Thus, we suggest that low-affinity interactions with Sm allow differentiation to an anergic B-2 cell, while higher-affinity interactions induce either developmental arrest or differentiation to B-1. Presumably, the affinity of the BCR for Sm also determines which cells differentiate to B-1, because, as shown in this work, low-affinity anti-Sm B cells do not become B-1. However, other features of anti-Sm binding may play a role, such as the ability to bind other autoantigens, such as DNA (17, 19, 22, 23).

Consistent with the idea that the fate of anti-Sm B cells is dependent on the strength of the tolerogenic signal, modulation of the BCR coreceptors CD19 and CD22 affects anti-Sm B cell differentiation in 2-12H Tg mice (24). Decreasing the strength of the tolerogenic signal by eliminating CD19 results in a detectable anti-Sm B-2 population in the spleens of 2-12H Tg mice, suggesting that cells that would normally be excluded from B-2 are now included (24). Conversely, increasing the tolerogenic signal results in an increase in the number of anti-Sm B-1 cells in 2-12H Tg mice (24).

That anergy is induced in low-affinity anti-Sm B cells while higher-affinity anti-Sm B cells are positively selected into the B-1 subset seems paradoxical. We have suggested that migration of B-1 cells to the peritoneum may decrease the exposure to self-Ag or that the expression of CD5, which is induced upon differentiation to a B-1 cell, raises the threshold of activation for these cells. Thus, selection into the B-1 subset may establish ignorance to Sm in nonautoimmune mice, which, like anergy, prevents the activation of anti-Sm B cells. Perhaps anti-Sm Abs have a protective function under certain conditions, and retaining some as functional B-1 cells has survival value. Alternatively, these cells could be selected into the B-1 subset as a consequence of a mechanism evolutionarily selected for B cells of other specificities that have a protective role, and the presence of anti-Sm B cells does no harm.

The elevated level of CD95, a proapoptosis receptor, suggests that low-affinity anti-Sm B cells will be eliminated upon T cell encounter. Elimination of anergic B cells via interaction with CD95 by T cells expressing CD95 ligand induces apoptosis of anergic anti-HEL B cells (39, 40), and engagement of CD95 is implicated in the regulation of anti-DNA B cells based on findings from CD95-deficient (lpr) mice (36, 41, 42, 43). Interestingly, while anergic anti-Sm B cells up-regulate CD95 and MHC class II, they show no evidence of up regulation of other cell surface proteins involved in B cell activation by T cells, such as CD40, CD80, CD86, and CD44 (Fig. 5Go and data not shown). This contrasts with anti-Sm B cells of 2-12H Tg mice that show up-regulation of CD40, CD44, and CD80, but not CD95 (44). Thus, low-affinity anti-Sm B cells may undergo apoptosis rather than activation upon interaction with T cells. This predicts that in the event of the loss of T cell tolerance to Sm, these anergic B-2 cells would be eliminated rather than activated. If so, then the activation of low-affinity anti-Sm B cells in lupus would require not only a loss of T cell tolerance, but also a change in the B cell to prevent cell death upon T cell interaction. This is consistent with the findings of others that there are defects in both B and T cells in MRL/lpr mice (20, 45, 46, 47). Such a change in the B cell could be the expression of an activation marker, especially B7.2, because signals provided by B7.2 lead to activation and proliferation of anergic B cells rather than deletion (48). Interestingly, splenic anti-Sm B cells of 2-12H/MRL/lpr mice, in which tolerance to Sm is lost, express elevated levels of B7.2 consistent with this possibility (44).

Although no general defects in central and peripheral tolerance in MRL/lpr (42, 49) or lpr-deficient mice (50) have been identified using Tg mouse models specific for the neo-Ags H2K and HEL, the regulation of lupus Ag-specific lymphocytes in autoimmune mice has shown different results. Anti-dsDNA Tg B cells in MRL/lpr mice no longer developmentally arrest and enter B cell follicles (41). Furthermore, Brard et al. (36) have demonstrated anti-ssDNA B cells that would otherwise be anergic can develop to ASC in MRL/lpr mice. Moreover, once activated, these cells acquired pathogenic specificities via somatic mutation and receptor editing. We have shown that tolerance to Sm is also lost in 2-12H MRL/lpr mice (44). The anti-Sm response is accelerated and increased in prevalence in these mice. Which anti-Sm B cells are involved in this response is not yet known, but the similarities between anti-ssDNA and low-affinity anti-Sm B cells suggest that it could involve the breakdown of anergy in low-affinity anti-Sm B cells. However, differentiation of anti-Sm B cells to B-1 cells is blocked in 2-12H MRL/lpr mice (44), suggesting that cells that would otherwise be induced to differentiate to B-1 may be diverted toward activation.

In summary, anergy, developmental arrest, and differentiation to B-1 are involved in the regulation of anti-Sm B cells. Which mechanism is used to regulate a given anti-Sm B cell depends at least in part on the affinity of their BCR. Low-affinity anti-Sm B cells appear to have a partial form of anergy, similar to anti-ssDNA B cells (7, 15). In addition, they have up-regulated the cell death receptor CD95, but not activation molecules important in B-T cell collaboration. Thus, anergic anti-Sm B cells appear poised to undergo cell death in the presence of cognate T cell help. The long half-life of anergic anti-Sm B cells suggests that they are an important population in the loss of tolerance to Sm in murine lupus.


   Acknowledgments
 
We gratefully acknowledge the Flow Cytometry Facility and the Cell and Molecular Physiology Imaging and Neuroscience Confocal Facility at University of North Carolina (Chapel Hill, NC). We also thank M. Ahinee Amamoo from the Biostatistics Facility at the Lineberger Comprehensive Cancer Center (University of North Carolina, Chapel Hill, NC) for her help with the statistical analysis, Dr. Garnett Kelsoe and the members of his laboratory at Duke University (Durham, NC) for their help with tissue sectioning, and P. Anne Wolthusen for her help with animal husbandry.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI29576, AI43587, and AI48085. Back

2 Address correspondence and reprint requests to Dr. Stephen H. Clarke, Department of Microbiology and Immunology, University of North Carolina, CB#7290, 804 MEJB, Chapel Hill, NC 27599. E-mail address: shl{at}med.unc.edu Back

3 Abbreviations used in this paper: BCR, B cell receptor; AP, alkaline phosphatase; ASC, Ab-secreting cell; BrdU, 5-bromo-2'-deoxyuridine; HEL, hen egg lysozyme; HSA, heat-stable Ag; MZ, marginal zone; Sm, Smith Ag; Tg, transgenic; V{kappa}8 Dbl Tg, 2-12H/V{kappa}8 double-Tg. Back

Received for publication July 13, 2001. Accepted for publication October 22, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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