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Impaired Intracellular Calcium Mobilization and NFATc1 Availability in Tolerant Anti-Insulin B Cells¹

Carlos A. Acevedo-Suárez^*,, Dawn M. Kilkenny, Martha B. Reich^* and James W. Thomas^2,*,

^* Department of Medicine, Department of Microbiology and Immunology, and Cell Imaging Shared Resource of Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, TN 37232

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
B lymphocytes that recognize soluble self-Ags are routinely found in normal individuals in a functionally inactive or anergic state. Current models indicate that this tolerant state is maintained by interactions with self-Ags that uncouple the BCR from downstream signaling pathways and increase levels of free calcium. Contrary to this expectation, B cells that harbor anti-insulin Ig transgenes (125Tg) are maintained in a tolerant state even though free calcium levels remain normal and tyrosine kinase substrate phosphorylation is preserved following BCR stimulation. Under basal conditions, intracellular levels of inositol 1,4,5-trisphosphate are increased and NFATc1 levels are reduced in 125Tg B cells. The 125Tg B cells are markedly impaired in their ability to mobilize calcium upon stimulation with ionomycin, and BCR-induced calcium mobilization from internal stores is decreased. In contrast, poisoning intracellular calcium pumps with thapsigargin increases calcium mobilization in 125Tg B cells. Changes in calcium signaling are accompanied by a failure of 125Tg B cells to translocate NFATc1 into the nucleus following stimulation with either anti-IgM or ionomycin. Thus, disassociation of BCR from multiple signaling pathways is not essential for maintaining tolerance in anti-insulin 125Tg B cells. Rather, BCRs that are occupied by autologous insulin deliver signals that induce changes in intracellular calcium mobilization and maintain tolerance by preventing activation of key transcription factors such as NFAT.

	Introduction

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Maintenance of unresponsiveness to self-Ags, i.e., immune tolerance, is essential for the prevention of autoimmune disease. During B cell development in the bone marrow, clonal deletion and receptor editing remove many autoreactive B cells from the repertoire (1, 2, 3). Engagement of the BCR by self-Ags with limited potential for aggregation does not cause deletion or editing; rather, it induces B cells to undertake a gene expression program that results in functional silencing and anergy (4). This process may render tolerant B cells susceptible to activation-induced death (5) or impair their ability to compete for follicular niches (6). The high prevalence of organ-specific autoimmune diseases (e.g., thyroiditis, type I diabetes, and pernicious anemia) and associated auto-Ab responses indicates that the tolerant state of B cells that recognize soluble self-Ags is highly vulnerable. Therefore, understanding how tolerance is maintained for self-Ags expressed at physiologic levels is key for developing strategies to prevent breaches in immune tolerance to specific auto-Ags.

The mechanisms that mediate tolerance for soluble self-proteins have been investigated largely using hen egg lysozyme (HEL)³ as a model neo-self-Ag (4, 5, 6, 7, 8, 9, 10). In this transgene model (anti-HEL/HEL), B cells that express high affinity (>10⁹ M) anti-HEL receptors in the presence of soluble HEL reduce surface IgM expression and lose key BCR-induced signaling responses. These tolerant B cells are refractory to anti-IgM stimulation as demonstrated by the reduction of tyrosine kinase substrate phosphorylation and the failure to mobilize NF-B. Anergic anti-HEL B cells also show an increase in basal intracellular calcium concentration ([Ca²⁺]_i) and fail to mobilize Ca²⁺ following receptor aggregation (8). A similar pattern of uncoupling of the BCR from downstream signaling pathways is observed in other tolerance models (11, 12). Consistent with the notion that BCR-derived signals are required for the completion of B lymphocyte development in the periphery (13), anergic B cells in many models of tolerance (anti-HEL/HEL, anti-dsDNA, and anti-Sm) share surface phenotypes of transitional B cells, suggesting that anergic cells undergo developmental arrest. This state is manifested by reduced CD23 expression on follicular B cells and by failure to generate marginal zone B cells (14, 15, 16, 17, 18). In these models, loss of tolerance is accompanied by a reversal of the developmental block (19, 20, 21, 22). Therefore, the reliance of tolerance on loss of BCR signals that alter development cannot be separated from its dependence on signals that maintain Ag-specific unresponsiveness, and it is not clear how tolerance is sustained in the absence of arrested development.

B cells that harbor anti-insulin Ig transgenes (125Tg) are well suited for the study of auto-Ag-induced signals that are conducive to tolerance without a block in B cell development. The 125Tg model differs significantly from other tolerance models in that its BCR is derived from a breach of tolerance in the primary immune response of a normal BALB/c mouse to insulin (23). In the anti-HEL model, the BCR is derived from hyperimmunization with a foreign Ag (7), and anti-DNA transgenics are derived from mice with established autoimmune disease (24). Tolerance for B cells harboring such high-affinity self reactivity is highly effective in normal immune systems in which their development is arrested (5, 7, 19). In contrast, although 125Tg B cells are anergic to BCR stimulation, they do not undergo developmental arrest (25); rather, they enter mature B cell subsets where they pose a risk by contributing to active immune responses. The presence of such B cells in the mature repertoire is consistent with the clinical observation that administration of autologous insulin routinely leads to the production of insulin Abs (26). Thus, a key issue for preventing autoimmunity is to understand how the threshold for tolerance is governed in self-reactive B cells that escape developmental arrest.

Analysis of BCR signaling in 125Tg B cells reveals that tyrosine kinase substrate phosphorylation remains coupled to the BCR following stimulation with anti-IgM and that basal [Ca²⁺]_i is not increased. The most striking features of anti-insulin B cells are impaired mobilization of Ca²⁺ from intracellular stores, reduced basal levels of NFATc1, and failure to induce NFATc1 mobilization following stimulation with anti-IgM or ionomycin. These findings contrast with other tolerance models such as anti-HEL, anti-DNA, and ARS/A1 and demonstrate that nonresponsiveness in mature B cells can be maintained without major alterations in BCR signaling. Our results indicate that anergy is not a fixed state but a continuum and, thus, not all anergic B cells share the same signaling profile. Furthermore, our data suggest that altered peripheral development is a feature of BCRs that are uncoupled from downstream signaling pathways.

	Materials and Methods

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Mice

The 125Tg mice used in this study harbor conventional anti-insulin Ig transgenes (Igµ only) derived from mAb 125 on a C57BL/6 background (backcross >27) and were generated and maintained as described previously (27, 28). B cells in 125Tg mice show no signs of allelic inclusion (25) and maintain high levels of allelic exclusion for long periods of time (>30 wk); 95% of B220⁺ lymphocytes express only IgM^a as revealed by staining with anti-IgM^a vs anti-IgM^b (27, 28). Nontransgenic (non-Tg) C57BL/6 mice (Taconic Farms) were used as controls. All mice were 8–12 wks old and were housed under specific pathogen-free conditions. All studies were approved by the Institutional Animal Care and Use Committee of Vanderbilt University (Nashville, TN).

Immunoblotting

Splenic B cells from non-Tg and 125Tg C57BL/6 mice were purified on magnetic beads (Miltenyi Biotec) and were routinely >90% pure as analyzed by anti-B220 (6B2; BD Pharmingen) Ab staining. Purified B cells were suspended in complete medium as described previously (25, 27) and stimulated (where specified) with 10 µg/ml F(ab')₂ goat anti-mouse µ-chain (Jackson ImmunoResearch Laboratories) or 1 µM ionomycin (Sigma-Aldrich) for the indicated times. Total cell lysates were prepared in lysis buffer containing 50 mM HEPES (pH 7.5) (Invitrogen Life Technologies), 5% glycerol (Invitrogen Life Technologies), 1% Triton X-100 (Sigma-Aldrich), 1 mM sodium orthovanadate (Sigma-Aldrich), 100 mM sodium chloride (Sigma-Aldrich), and protease inhibitors (Roche Applied Science). Cytoplasmic and nuclear extracts were separated as previously described (29, 30). Total protein loaded was verified by BCA assay (Pierce). Samples were analyzed by immunoblotting using an ECL system (Amersham Biosciences). Ab reagents purchased from Santa Cruz Biotechnology were reactive with phospho-tyrosine (clone pY99), phospho-Lyn (polyclonal), Lyn (polyclonal), phospholipase C (PLC2) (polyclonal), NFATc1 (clone 7A6), and G (polyclonal) (loading control). Abs reactive with phospho-Syk (polyclonal), Syk (polyclonal), and phospho-PLC2 (polyclonal) were obtained from Cell Signaling Technology. Anti-IgM Ab (polyclonal) was purchased from Chemicon.

Ca²⁺ mobilization assay

Purified splenic B cells from non-Tg and 125Tg mice were suspended in HBSS (Invitrogen Life Technologies) containing 50 mM HEPES (pH 7.5) and plated on poly-D-lysine-coated glass bottom microwell dishes (MatTek) for 30 min at 37°C. Cells were loaded with the cell-permeant, Ca²⁺-sensitive dye fura 2-AM (5 µM) (Molecular Probes) for 30 min at room temperature and then washed in HBSS. For some experiments, fura 2-AM-loaded cells were suspended initially in Ca²⁺-free HBSS (Cambrex) containing 2 mM EGTA (Sigma-Aldrich) that was supplemented with 2 mM CaCl₂ (Sigma-Aldrich) after 5 min. The dishes were mounted on an inverted Nikon TE300 microscope enclosed in a humidified Plexiglas chamber maintained at 37°C with an atmosphere of 5% CO₂. Images were collected using a x40, 1.3 numerical aperture, oil immersion Plan Fluor objective lens and a side-mounted CoolSNAP_HQ camera. Fluorescence was monitored using dual excitation wavelengths (340/380 nm) and a single emission wavelength (510 nm). Fluorescence at 340 nm indicates dye bound to Ca²⁺, whereas that at 380 nm corresponds to free dye. Basal readings were taken for 45 s before stimulation. Metamorph imaging software (Universal Imaging) was used for automated collection of images at either 1 or 5 s intervals over a 5–10 min incubation period. During this time, cells maintained healthy morphology. Average fluorescence measurements were determined using Metamorph software. Data are expressed as the ratio of bound:free fura 2-AM fluorescence intensities.

Inositol 1,4,5-trisphosphate (IP₃) production analysis

Purified splenic B cells (2 x 10⁶) from non-Tg and 125Tg mice were suspended in 50 µl of complete medium and stimulated with 10 µg/ml F(ab')₂ goat anti-mouse µ-chain at 37°C for the times indicated. IP₃ production was determined by Biotrak IP₃ assay system (Amersham Biosciences). Data from three independent experiments are expressed as the ratio of IP₃ levels at each time point over basal IP₃ levels in non-Tg B cells. Error bars represent SEM (Fig. 5).

View larger version (17K): [in this window] [in a new window]   FIGURE 5. IP3 metabolism is altered in anti-insulin 125Tg B cells. Non-Tg and 125Tg B cells were stimulated with 10 µg/ml F(ab')2 goat anti-mouse IgM for the indicated times. IP3 production was determined by Biotrak assay. Data are expressed as relative IP3 levels at each time point over basal IP3 levels in non-Tg B cells.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Anti-insulin 125Tg B cells mature normally but are maintained in an anergic state by autologous insulin occupying their BCR (25, 27). To examine the BCR-derived responses of B lymphocytes that have been rendered tolerant by exposure to physiologic concentrations of insulin in vivo, protein p-Tyr was examined following stimulation with 10 µg/ml anti-IgM. This dose is in the recognized optimal range for responses of normal B cells and was used in studies of anergic B cells in other tolerance models to unmask signaling defects in those systems (10, 12). The kinetics of the p-Tyr response was compared using Western blotting (Fig. 1, top) on control (lanes 1–4) and 125Tg (lanes 5–8) B cells. Basal phosphorylation is similar in both non-Tg and 125Tg B cells (compare lanes 1 and 5 in Fig. 1). Within 1 min of stimulation, multiple proteins are phosphorylated in control and 125Tg B cells (Fig. 1, lanes 2 and 6). Phosphorylation of Lyn and Syk (arrows) was confirmed in separate experiments (not shown). Similarly, there was no difference between non-Tg and 125Tg B cells in phosphorylated phospholipase C (PLC) 2 either basally or following BCR stimulation (Fig. 2). The 125Tg B cells have a relative increase of 1.2 for IgM H chain (not shown). Although the overall p-Tyr response is more robust and of longer duration in 125Tg (Fig. 1, lanes 6–8) than in non-Tg B cells (Fig. 1, lanes 2–4), a longer exposure of the blot (Fig. 1, middle panel) shows that the same molecular mass proteins are targeted by BCR signaling in control and 125Tg B cells. These data are highly reproducible and clearly show that the p-Tyr response is not impaired in tolerant anti-insulin B cells.

View larger version (68K): [in this window] [in a new window]   FIGURE 1. Protein p-Tyr is highly active in anti-insulin 125Tg B cells following stimulation with anti-IgM. Non-Tg (lanes 1–4) and 125Tg (lanes 5–8) B cells were stimulated with 10 µg/ml F(ab')2 goat anti-mouse IgM for the indicated times and subjected to immunoblotting (4 µg of total protein per lane) with anti-phospho-tyrosine-specific Ab and anti-G Ab (loading control). Molecular mass markers are indicated in the far left column. Top panel shows short exposure, and middle panel shows long exposure.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. PLC2 phosphorylation (p-PLC2) is normal in 125Tg B cells. Non-Tg (lanes 1–4) and 125Tg (lanes 5–8) B cells were stimulated with 10 µg/ml F(ab')2 goat anti-mouse IgM for the indicated times and subjected to immunoblotting (4 µg of total protein per lane) with Abs to phospho-PLC2, PLC2, and G (loading control). p-PLC2 levels were normalized to the loading control and are indicated immediately below the blot.

Activation of Ag receptors on immune cells normally leads to a rapid change in [Ca²⁺]_i via several mechanisms, including Ca²⁺ release from internal stores and Ca²⁺ influx across the plasma membrane (9, 31). In the tolerance models studied to date, anergic B cells are characterized by an increase in basal [Ca²⁺]_i and by impaired Ca²⁺ mobilization following BCR stimulation (5, 6, 11, 12, 32). Therefore, the ability of non-Tg and 125Tg B cells to mobilize Ca²⁺ upon stimulation was measured in fura 2-AM-loaded cells (Fig. 3). In all experiments, the relative [Ca²⁺]_i under basal conditions (45 s before treatment), was not measurably different between control and 125Tg B cells (n = 12). Following stimulation with 10 µg/ml anti-IgM (Fig. 3, third panel from the top; indicated by arrow), control and 125Tg B cells generated a Ca²⁺ flux of similar magnitude and duration. At suboptimal doses of anti-IgM (1 µg/ml), non-Tg B cells had a slightly higher Ca²⁺ response than 125Tg B cells (Fig. 3, second panel from the top). Non-Tg B cells treated with 5 µM ionomycin (Fig. 3, bottom panel) generated a steep rise in intracellular Ca²⁺ immediately following stimulation that was sustained for the duration of the experiment. The magnitude of the Ca²⁺ response to ionomycin in non-Tg B cells was greater than that evoked following BCR cross-linking. In striking contrast, when tolerant 125Tg B cells were treated with 5 µM ionomycin the magnitude of the Ca²⁺ flux was reduced compared with that of non-Tg B cells (Fig. 3, bottom panel). This response, however, was also sustained for the duration of the experiment. The Ca²⁺ response of 125Tg B cells was also impaired at lower doses of ionophore (not shown). These results indicate that the capacity of tolerant anti-insulin B cells to flux Ca²⁺ in response to treatment with either ionophore or 1 µg/ml anti-IgM is impaired even though their ability to mobilize Ca²⁺ following 125Tg BCR cross-linking with 10 µg/ml anti-IgM appears to be preserved.

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Ca2+ mobilization in 125Tg B cells is impaired following treatment with ionomycin and suboptimal doses of anti-IgM. Non-Tg and 125Tg B cells were loaded with fura 2-AM, washed, and resuspended in HBSS. Basal fluorescence readings were taken for 45 s before stimulation (solid arrow) with 0.1 (top panel), 1 (second panel), and 10 µg/ml (third panel) F(ab')2 goat anti-mouse IgM or 5 µM ionomycin (bottom panel).

Increases in intracellular Ca²⁺ arise from two sources, release from intracellular stores in the ER, and through channels in the cell membrane that permit Ca²⁺ to move from the extracellular environment into the cell (9, 31). Therefore, the impaired Ca²⁺ response of 125Tg B cells could be the result of diminished Ca²⁺ mobilization across the ER, failure to induce Ca²⁺ influx from extracellular sources across the plasma membrane, or both. To distinguish between these possibilities, we measured the Ca²⁺ response of non-Tg and 125Tg B cells following stimulation in Ca²⁺-free buffer that was subsequently supplemented with 2 mM CaCl₂. In this way, the initial Ca²⁺ release from internal stores can be separated from the ensuing Ca²⁺ influx across the plasma membrane. When control B cells were treated with 10 µg/ml anti-IgM in the absence of Ca²⁺ (Fig. 4, top panel; solid arrow), an increase in fura 2-AM fluorescence indicative of free Ca²⁺ released from the ER was observed. Following the addition of Ca²⁺ to the medium (open arrow), [Ca²⁺]_i increased further and was sustained for the duration of the experiment. In contrast, anti-IgM stimulation of 125Tg B cells in the absence of Ca²⁺ (Fig. 4, top panel) induced a limited Ca²⁺ response from the ER that was less than that of control B cells. However, upon addition of Ca²⁺ to the medium, [Ca²⁺]_i increased to comparable levels in non-Tg and 125Tg B cells.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Impaired Ca2+ release localizes to the ER in tolerant anti-insulin B cells. Non-Tg and 125Tg B cells were loaded with fura 2-AM, washed, and resuspended in Ca2+-free HBSS containing 2 mM EGTA. Basal fluorescence readings were taken for 45 s before stimulation (solid arrow) with 10 µg/ml F(ab')2 goat anti-mouse IgM (top panel), 1 µM ionomycin (middle panel), or 0.2 µM thapsigargin (bottom panel). Ca2+-free HBSS was supplemented with 2 mM CaCl2 after 5 min (open arrow), and fluorescence was monitored for an additional 5 min.

One mechanism regulating ER Ca²⁺ release is the activity of the sarco-endoplasmic Ca²⁺-ATPase (SERCA), which mediates the reuptake of Ca²⁺ from the cytoplasm into the ER. In the absence of Ca²⁺, the ATPase activity of SERCA is low, leading to a slow leak of Ca²⁺ from intracellular stores (33, 34). In addition, inhibition of SERCA activity by the drug thapsigargin results in an increase in [Ca²⁺]_i that reflects Ca²⁺ release from ER stores. Accordingly, the Ca²⁺ response of non-Tg and 125Tg B cells to thapsigargin was examined. Treatment with thapsigargin induced greater Ca²⁺ release from the ER in 125Tg B cells than in control B cells (Fig. 4, bottom panel). This increased responsiveness to thapsigargin treatment generates a small but consistently higher level of extracellular Ca²⁺ influx in 125Tg B cells relative to control B cells when Ca²⁺ is returned to the medium. These data, together with the impaired sensitivity to anti-IgM and ionomycin treatment, suggest that increased SERCA activity in 125Tg B cells maintains normal [Ca²⁺]_i in tolerant B cells by altering the threshold of ER responses.

IP₃ metabolism is altered in tolerant anti-insulin B cells

BCR-derived signals lead to the production of IP₃ which, upon binding to its receptor (IP₃R) in the ER, induces the release of stored Ca²⁺ (35). Therefore, IP₃ generation was measured in non-Tg and 125Tg B cells (Fig. 5). The data reveal that basal IP₃ levels are elevated 3.5-fold in anti-insulin 125Tg B cells compared with control B cells. Upon BCR engagement for 1 min, generation of IP₃ is increased 2.5-fold over basal levels in control B cells but only 12.5% in 125Tg B cells. After 3 min of BCR stimulation, non-Tg B cells increased IP₃ production 3-fold over basal levels, whereas 125Tg B cells increased IP₃ generation only 20%. These data indicate that basal IP₃ production is elevated in anti-insulin 125Tg B cells and that the ability of anti-IgM to increase BCR-induced generation of IP₃ is impaired in 125Tg B cells relative to non-Tg B cells.

Basal NFATc1 levels are reduced in anti-insulin 125Tg B cells

The transcription factor NFAT is a key downstream mediator of BCR-dependent signaling that is dependent upon Ca²⁺ mobilization. In T cells induced to become anergic by prolonged ionomycin treatment, the genetic regulation of anergy appears to be governed by the availability of different NFATs (36). Therefore, basal NFATc1 levels were analyzed by Western blotting (Fig. 6) in total extracts prepared from freshly isolated B cells from individual non-Tg (lanes 1–4) and 125Tg (lanes 5–8) mice. B cells from non-Tg mice express significantly higher (3.6-fold) NFATc1 levels than those of mice harboring 125Tg (1.74 ± 0.81 vs 0.49 ± 0.25, p = 0.025 by Student’s t test). These data show that NFATc1 expression is reduced in anti-insulin B cells and suggest that NFAT availability might play a role in regulating tolerance in 125Tg B cells.

View larger version (38K): [in this window] [in a new window]   FIGURE 6. Basal NFATc1 levels are reduced in anti-insulin 125Tg B cells. Total cell lysates were prepared from freshly isolated B cells from individual non-Tg (lanes 1–4) and 125Tg (lanes 5–8) mice and were subjected to immunoblotting (4 µg of total protein per lane) using anti-NFATc1 and anti-G Abs (loading control). NFATc1 levels were normalized to the loading control and are indicated immediately below the blot.

NFAT activation was analyzed by Western blotting following stimulation of non-Tg and anti-insulin 125Tg B cells with anti-IgM. Translocation of NFATc1 from the cytoplasm into the nucleus was readily detected following BCR cross-linking in non-Tg B cells (Fig. 7, lanes 1–3; 90–96% depletion from cytoplasm). In contrast, NFATc1 translocation was not detectable above basal levels in 125Tg B cells after 30 min of anti-IgM stimulation (Fig. 7, lanes 5–7; 17–42% depletion from cytoplasm). Treatment with ionomycin produced a dramatic depletion of NFATc1 from the cytoplasm of control B cells (> 98%; compare lane 1 to lane 4 in Fig. 7). However, in 125Tg B cells ionomycin treatment resulted in a markedly reduced translocation of cytoplasmic NFATc1 into the nucleus (50% depletion from cytoplasm; compare lane 5 to lane 8 in Fig. 7) relative to control B cells. The inability to initiate nuclear translocation of NFATc1 in 125Tg B cells is consistent with the altered intracellular mobilization of Ca²⁺ that we find using these same stimuli in tolerant 125Tg B cells.

View larger version (67K): [in this window] [in a new window]   FIGURE 7. Anti-insulin 125Tg B cells fail to activate NFATc1 following BCR or ionomycin stimulation. Non-Tg (lanes 1–4) and 125Tg (lanes 5–8) B cells were treated with either 10 µg/ml F(ab')2 goat anti-mouse IgM (lanes 2–3, and 6–7) for the indicated times or with 1 µM ionomycin (lanes 4 and 8) for 30 min. Cytoplasmic (4 µg of total protein per lane) and nuclear (2 µg of total protein per lane) extracts were prepared and subjected to immunoblotting using anti-NFATc1 and anti-G Abs (loading control). NFATc1 levels were normalized to the loading control and are indicated immediately below each blot.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

Although basal [Ca²⁺]_i is not increased in 125Tg B cells, Ca²⁺ signaling pathways play a key role in maintaining tolerance in 125Tg B cells. The data show that anti-insulin B cells have increased basal IP₃ levels (Fig. 5), impaired Ca²⁺ mobilization from internal stores following optimal BCR stimulation (Fig. 4), and both impaired NFATc1 production and impaired NFATc1 activation (Figs. 6 and 7). These findings indicate that 125Tg BCR occupancy by insulin generates signals that can sustain tolerance without altering peripheral development and without profound changes in BCR-associated tyrosine kinase activity. The more severe impairments in BCR signaling in other models of tolerance, including anti-HEL, are likely reflected in the B cell development block observed in these systems. A critical difference in tolerance between 125Tg B cells and B lymphocytes in other models such as anti-HEL/HEL resides in the way Ag availability is regulated. In the anti-HEL model auto-Ag levels are fixed, whereas for anti-insulin B cells Ag levels fluctuate according to the physiologic regulation of glucose concentration. Thus, hormone removed from circulation by binding to anti-insulin BCRs is quickly replaced so that Ag is continuously available. Our findings that most 125Tg BCRs are occupied by insulin (27) are consistent with this process. Thus, our data indicate that anergy is not a fixed state but a continuum of alterations that result from quantitative and qualitative changes in BCR signaling.

A previously unrecognized feature of tolerance that we find in anti-insulin B cells is impaired Ca²⁺ release from intracellular stores (Fig. 4). One indication of impaired Ca²⁺ mobilization in 125Tg B cells is their blunted response to ionomycin (Fig. 3), an agent commonly used as a positive control in Ca²⁺ studies. Previous work in T cells selected for signaling defects found that resistance to ionomycin-induced intracellular Ca²⁺ elevation can be due to failure to evoke Ca²⁺ mobilization across the plasma membrane (37). In contrast, experiments using ionomycin or anti-IgM in the absence of extracellular Ca²⁺ reveal that impaired Ca²⁺ release in 125Tg B cells is due to a failure to mobilize Ca²⁺ from intracellular stores (Fig. 4). Unlike the response to treatment with 5 µM ionomycin, the total Ca²⁺ response of 125Tg B cells is not markedly impaired following stimulation with 10 µg/ml anti-IgM (Fig. 3). This outcome may reflect the numerous signals generated by extensive BCR cross-linking compared with ionomycin, which may directly target the ER (38). An attenuated ER response to anti-IgM is unmasked when the availability of extracellular Ca²⁺ is restricted (Fig. 4, top panel). This finding suggests that, under normal Ca²⁺ conditions, a large Ca²⁺ influx across the plasma membrane obscures an impaired ER Ca²⁺ response of 125Tg B cells following treatment with 10 µg/ml anti-IgM (Fig. 3, top panel). Only a small intracellular signal (as revealed in Fig. 4, top panel) is needed to trigger this influx of extracellular Ca²⁺, which buries the small peak corresponding to release of intracellular Ca²⁺. These observations demonstrate that processes tightly regulating intracellular Ca²⁺ signals are connected to the tolerant state in anti-insulin B cells. Consistent with this idea, recent studies suggest that persistent BCR occupancy by self-Ag is required for maintaining impaired Ca²⁺ mobilization and the anergic state in an Ig transgenic model in which B cells specific for a hapten (arsonate) cross-react with an auto-Ag (39). Our data further indicate that the tolerant state of anti-insulin B cells can be overcome, consistent with the loss of tolerance to insulin as occurs in autoimmune (type 1) diabetes and after insulin therapy.

In B cells, binding of IP₃ to its receptor on the ER membrane is the primary means of inducing Ca²⁺ release following BCR stimulation. Ca²⁺ and IP₃ regulate the Ca²⁺ channel activity of IP₃R in a biphasic manner so that elevated levels of either Ca²⁺ or IP₃ in the cytoplasm will also inhibit Ca²⁺ release from ER stores (40, 41, 42). In addition, removal of IP₃ is an active process (43, 44, 45) that may be impaired in the tolerant state. Thus, the high basal levels of IP₃ found in 125Tg B cells (Fig. 5) are likely to play a role in attenuating ER Ca²⁺ release in response to ionomycin and following anti-IgM stimulation. In forms of tolerance induced by high affinity interactions such as anti-HEL/HEL, elevated levels of free intracellular Ca²⁺ may play a similar role in dampening Ca²⁺ responses (9, 10). Understanding how BCR occupancy with paucivalent ligands, such as insulin, lead to sustained IP₃ levels is an important issue for future studies.

Further insight into the mechanisms maintaining Ca²⁺ homeostasis in 125Tg B cells is provided by the use of thapsigargin to inhibit SERCA activity (Fig. 5, bottom panel). More Ca²⁺ is released in response to thapsigargin treatment by 125Tg B cells than by non-Tg B cells, suggesting that SERCA expression and/or activity is augmented in anergic B cells. Increased SERCA activity may explain why basal Ca²⁺ levels are normal in 125Tg B cells rather than elevated as in other tolerance models. Because insulin binding to the 125Tg BCR does not extensively cross-link surface IgM, it is likely that under basal conditions there is only a modest increase in ER Ca²⁺ release that is compensated for by higher total SERCA activity. Therefore, the increased [Ca²⁺]_i observed in the high affinity tolerance models may reflect the inability of those cells to compensate for the augmented Ca²⁺ load induced by more intense BCR stimulation.

Our findings of normal levels of p-PLC2 (Fig. 2) are unexpected given the elevated IP₃ production in 125Tg B cells. However, the phosphorylation status of PLC does not necessarily reflect all of its activity (45, 46). Thus, it is possible that fine differences in the activation state of PLC2, not revealed by phosphorylation, contribute to the impaired Ca²⁺ response of 125Tg B cells. Studies in PLC2-deficient B cells have indicated that lipase-independent PLC2 activities may have a role in BCR-dependent Ca²⁺ responses (47). Furthermore, Cbl proteins, known to negatively regulate BCR signaling in part by directing Syk kinase degradation (48), can also target PLC2 (49), suggesting that protein turnover and/or recruitment could be altered in tolerant B cells.

A feature of anergy in 125Tg B cells that is shared with other tolerance models is the dysregulated activation of NFAT. The impaired Ca²⁺ response of 125Tg B cells is associated with increased basal levels of nuclear NFATc1 and with failure to induce further translocation of NFATc1 from the cytoplasm into the nucleus following stimulation with either anti-IgM or ionomycin (Fig. 7). Although a similar outcome is also observed in the anti-HEL system (9, 10), the block in NFATc1 translocation, especially in response to ionomycin, is much more severe in anti-insulin 125Tg cells. Furthermore, no defect in total NFAT levels were reported in tolerant anti-HEL B cells. In contrast, we have found a significant reduction (3.6-fold) in total NFATc1 expression in 125Tg B cells (Fig. 6), suggesting that the amount of NFAT available for induction might be limited in these cells. This finding suggests that the inability of anti-insulin B cells to proliferate in response to anti-IgM stimulation in vitro (25) may be linked to impairments of nuclear NFAT activity. Studies in other systems suggest that NFAT proteins play a key role in regulating cell cycle progression (50, 51). In addition, in T cells induced to become anergic by prolonged ionomycin treatment, the genetic regulation of anergy appears to be governed by the availability of different nuclear NFATs (36). Such similarities in diverse systems suggest that altered expression and induction of NFATs, and possibly other transcription factors, may play a common role in silencing autoimmune responses.

It is possible that the hormone receptor for insulin (InsR) could have a role in insulin immunity and tolerance. However, we do not detect differences in 125Tg B cell responses when either hormonally active or inactive insulin is used (not shown). In addition, previous studies have shown that mAb125 does not recognize InsR-bound insulin (52) and that resting T and B cells do not express InsR (53). However, both BCR and InsR traffic in clathrin vesicles; thus, it remains possible that some interactions in the endosomal pathway generate signals during intracellular processing. Studies are in progress to explore this possibility.

Our findings are consistent with a model (Fig. 8) in which continuous BCR occupancy by insulin alters normal basal signaling and triggers compensatory mechanisms that lead to impaired Ca²⁺ mobilization (Fig. 4) and decreased NFAT activity (Fig. 7). This attenuated Ca²⁺ response is not associated with the uncoupling of 125Tg BCR from downstream signaling pathways, because overall p-Tyr is intact (Fig. 1). Instead, signaling from an occupied BCR may lead to heightened SERCA activity (Fig. 4) and contribute to the impaired Ca²⁺ response by elevating basal IP₃ levels (Fig. 6) that may further impair Ca²⁺ release. Another possibility is that the decreased ER Ca²⁺ response in 125Tg B cells might be regulated at the level of the IP₃R, thus altering the action of Ca²⁺ channels in the ER. The observed sustained phosphorylation of target proteins following BCR cross-linking in 125Tg B cells (Figs. 1 and 2) may result directly or indirectly in the persistent activation of an inhibitor of the Ca²⁺ channel activity of the IP₃R. Consistent with this idea, recent data show that BCR stimulation may allow Lyn (a positive and negative regulator of BCR signaling) to modulate Ca²⁺ mobilization from internal stores and across the plasma membrane (54, 55, 56). Studies of these mechanisms are in progress to examine the role of the IP₃R and BCR-induced phosphorylation in the impaired Ca²⁺ response of 125Tg B cells.

View larger version (31K): [in this window] [in a new window]   FIGURE 8. Model for Ca2+ signaling in tolerant anti-insulin B cells. Persistent BCR occupancy by insulin alters normal basal signaling and triggers compensatory mechanisms that lead to impaired Ca2+ mobilization and decreased NFAT activity. Engagement of the 125Tg BCR with insulin leads to increased basal levels of IP3 and nuclear NFATc1 while preserving BCR-dependent tyrosine kinase activity. The increased basal signals may augment SERCA activity, further impairing intracellular Ca2+ mobilization. Changes in basal signaling attenuate ER Ca2+ release following stimulation with anti-IgM or ionomycin and result in the failure of NFATc1 translocation into the nucleus above basal levels. PIP2, phosphatidylinositol 4,5-bisphosphate.

	Acknowledgments

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by National Institutes of Health Grants AI051448 and AR46732 (to J.W.T.). The Cell Imaging Shared Resource of Vanderbilt-Ingram Cancer Center is supported in part by National Institutes of Health Grant CA068485.

² Address correspondence and reprint requests to Dr. James W. Thomas, Department of Medicine, Vanderbilt University, Nashville, TN 37232-2681. E-mail: james.w.thomas{at}vanderbilt.edu

³ Abbreviations used in this paper: HEL, hen egg lysozyme; [Ca²⁺]_i, intracellular Ca²⁺ concentration; ER, endoplasmic reticulum; InsR, hormone receptor for insulin; IP₃, inositol 1,4,5-trisphosphate; p-Tyr, tyrosine phosphorylation; PLC2, phospholipase C2; SERCA, sarco-endoplasmic Ca²⁺-ATPase; 125Tg, anti-insulin Ig H and L chain transgene; non-Tg, nontransgenic; IP₃R, IP₃ receptor.

Received for publication January 19, 2006. Accepted for publication June 6, 2006.

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