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Tumor Immunology Program, German Cancer Research Center, Im Neuenheimer Feld 280, 6900 Heidelberg, Germany; Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria 3050 Australia; || Department of Medicine, Microbiology and Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri 63198; and ¶ Department of Immunology, University of Colorado Health Sciences Center, Denver, Colorado 80220
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(anti-Kk,b) Ig Tg mice to H-2b mice or to mice expressing transgene-driven Kb in the periphery. In 3–83µ/bcl-2 Tg mice, deletion of autoreactive B cells induced by peripheral Kb antigen expression in the liver (MT-Kb Tg) or epithelia (KerIV-Kb Tg), was partly or completely inhibited, respectively. Furthermore, Bcl-2 protected peritoneal B-2 B cells from deletion mediated by acute antigen exposure, but this protection could be overcome by higher antigen dose. In contrast to its ability to block peripheral self-tolerance, Bcl-2 overexpression failed to inhibit central tolerance induced by bone marrow antigen expression, but instead, enhanced the receptor editing process. These studies indicate that apoptosis plays distinct roles in central and peripheral B cell tolerance.
Apoptosis is an essential element in the development and homeostasis of many tissues. In the immune system, a number of important processes are regulated through the control of cell death and survival (1–4). Among these processes is immunological tolerance in which encounter with ligands that signal through antigen receptors affect the cell's subsequent survival (5, 6). The lifespans of lymphocytes in vivo vary widely, from 1–2 d to many weeks (7). Self-antigen can shorten the lifespan of reactive B cells in a number of ways: by promoting cell death through developmental arrest (8, 9), by increasing cell turnover (10, 11), by putting cells at a competitive disadvantage with nonautoreactive cells for unknown resources (12), by making the cells sensitive to Fas ligand–mediated killing by T cells (13), or apparently through direct induction of apoptosis (11, 14–18).
One regulator of lymphocyte survival is the Bcl-2 protein (1), which is highly expressed in long-lived lymphocytes and is poorly expressed in cells destined to turn over rapidly (19–26). Bcl-2, and the closely related Bcl-xL (27), are key protein regulators of apoptosis. Overexpression or inappropriate expression of these proteins can play a role in lymphoma development and can allow the continued survival of cells that would otherwise be lost through apoptosis (28–36). However, some forms of apoptosis, including Fas-mediated killing of certain lymphoid cells (37), are not inhibitable by Bcl-2. An important physiological role for Bcl-2 is evident from the phenotype of bcl-2–deficient mice, which manifest a catastrophic apoptotic loss of mature lymphocytes (38, 39).
The ability of Bcl-2 to block B cell tolerance has been studied in several systems in which ligands that bind to the B cell receptor (BCR)1 can stimulate cell death in vivo or in vitro (9, 15, 33, 40–48). In some cases, these data have been contradictory, suggesting that the ability of Bcl-2 overexpression to block tolerance-mediated apoptosis may be contingent upon the precise quality of the BCR-mediated signal and perhaps other signals that may differ in certain experimental systems. In the well-characterized surface Ig+ B lymphoma WEHI-231, cross-linking of the BCR with antiimmunoglobulin antibody leads to cell death (for review see reference 18). In some studies (43, 44), but not others (40), transfection of bcl-2 expression constructs protected the cells from BCR-mediated cell death. When transgenic (Tg) mice with enforced B cell overexpression of Bcl-2 were analyzed for B cell tolerance, bone marrow tolerance was perturbed in one study (9), but not in another (46), whereas peripheral tolerance was inhibited (15, 46). Similarly, germinal center B cells have been shown to be sensitive to killing induced by acute ligation of sIg, and this death could be inhibited by enforced Bcl-2 expression in one study (48), but not in another (47). These studies and others focusing on T cell tolerance (3, 4) have suggested that the ability of Bcl-2 to protect autoreactive lymphocytes from cell death may be contingent upon many factors, including the developmental stage of the lymphocyte as well as the nature of the antigen stimulus.
Resting B cells encountering tissue-specific membrane-bound antigen in the periphery are eliminated, suggesting that in this case, peripheral tolerance is mediated by an apoptotic process (14). In contrast, it has been shown in several systems that immature B cells encountering membrane-bound or nuclear self-antigens in the bone marrow are blocked in their development (8, 9) and either undergo receptor editing (49, 50) or are eliminated (50–52). Our recent finding that in vitro tolerance induction of immature B cells stimulates intense receptor editing with little immediate cell death (53, 54) suggests that antigen-induced programmed cell death makes only a minor contribution to central tolerance. Assuming that Bcl-2 is a key cell death regulator in B cells, this model predicts that enforced Bcl-2 expression can block clonal elimination of mature, but not immature, self-reactive B cells. To test this, peripheral and central tolerance in 3–83µ
Mouse Typing.
Antibodies, Immunofluorescence, Cell Lines, and Intraperitoneal Injection Assays.
Cell Culture.
5-bromo-2'-deoxyuridine Incorporation Assay. (anti–H-2k,b) Ig Tg mice were compared to that occurring in 3–83µ/Eµ–bcl-2-22 double-Tg mice, in which Bcl-2 is constitutively expressed in B-lineage cells.
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Mice.
MT-Kb mice, in which Kb antigen expression is directed to hepatocytyes by the sheep metallothionein promoter (55), and KerIV-Kb mice, in which Kb is expressed in epithelial cells under the control of the keratin IV promoter (56), were bred several generations onto the B10.D2 background (H-2d) before breeding with other Tg or H-2 congenic mice. 3–83µ Tg mice, which express IgM and IgD forms of the 3–83 antibody, have been described (14). 3–83µ mice were backcrossed a minimum of 10 times onto the B10.D2 background. C57BL/6(H-2b), C3H-H-2o2/SfSn (C3H.OH: KdDk), and B10.D2/nSnJ (H-2d) mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice overexpressing the human bcl-2 gene in B-lineage cells were Tg lines Eµ–bcl-2-22 (34) and bcl-2-Ig NL (36). These mice were backcrossed a minimum of three generations to B10.D2 and subsequently bred with 3–83µ Tg mice. The phenotypes of mice bearing either of the bcl-2 transgenes were indistinguishable in our study. Unless otherwise stated, the experiments described made use of the Eµ–bcl-2-22 line. The 3–83µ/RAG-1 knockout mice (57) were bred with Eµ–bcl-2-22 Tg mice. F2 crosses were set up to generate bcl-2/3–83µ/RAG-1–/– mice with all transgenes hemizygous except where noted. All mice were bred and maintained under specific pathogen-free conditions in the animal care facility at the National Jewish Medical and Research Center (NJMRC, Denver, CO).
PCR typing for 3–83µ, Kb, and bcl-2 transgenes was as described previously (14). RAG-1–deficient mice were identified by their lack of T cells in flow cytometric analysis of peripheral blood.
Preparation of lymphoid cells, antibody reagents, fluorochrome conjugation, flow cytometry analysis, cell lines, and intraperitoneal injection assay methods were as previously described (58). In brief, the A/J splenocyte x Sp2/0 hybridoma 4549 (which expresses Kk) and Sp2/0 (H-2d) parent hybridoma were washed and resuspended at either 5 x 107 or 5 x 108 cells/ml in PBS and injected intraperitoneally into 3–83µ/H-2d or 3–83µ/H-2d/bcl-2 Tg mice. Approximately 16 h later, the peritoneal cells were harvested with 10 ml HBSS, washed twice, and stained for the presence of Id-reactive antibodies.
Spleen or lymph node cells were cultured in RPMI-1640 medium supplemented with 10% FCS and 50 µM 2-mercaptoethanol ± 50 µg/ml LPS (Sigma Chemical Co., St. Louis, MO). The cell concentration was adjusted depending on the number of 3–83 B cells in the organ according to the following guidelines: 106 cells/ml for H-2d 3–83 Tg, H-2d bcl-2/3–83 double-Tg, and bcl-2/3–83µ/KerIV-Kb triple-Tg mice; and 3–5 x 106 cells/ml for H-2b 3–83 Tg, H-2b bcl-2/3–83µ, 3–83/ MT-Kb, 3–83/KerIV-Kb double-Tg, and bcl-2/3–83µ/MT-Kb triple-Tg mice. In some experiments, lymph node and spleen cells were T cell depleted with anti-Thy1 antibodies and rabbit complement and cultured at 5 x 105 cells/ml. Culture supernatants were harvested on days 3 and 7 after initiation of culture and Ig concentrations determined by ELISA, as described (14).
5-bromo-2'-deoxyuridine (BrdU; Sigma Chemical Co.) was provided in filtered, deionized drinking water at a concentration of 1 mg/ml for 7 d. Staining for the incorporation of BrdU was described previously (59).
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Enforced Bcl-2 Expression Blocks B Cell Deletion Induced by Acute Antigen Administration at Low Dose, but Not at High Dose.
The 3–83µ Ig transgene encodes a BCR that is reactive to a number of MHC class I alloforms including Kk, Dk, and Kb, but fails to bind to H-2d; thus, 3–83µ/H-2d mice contain a virtually monoclonal B cell population bearing the 3–83 BCR and are called nondeleting (ND)Tg mice (14). To probe the ability of enforced Bcl-2 expression to block tolerance to acute intraperitoneal antigen challenge, we injected NDTg and NDTg/bcl-2 mice with antigen-bearing cells, a protocol that stimulates rapid apoptosis of fully mature antigen-specific peritoneal B cells (15, 16). Injection of hybridoma cells bearing the high-affinity Kk antigen consistently led to massive loss (
80%) of the 3–83µ peritoneal B cells over a 16-h period, whereas injection of control H-2d cells did not (Fig. 1). At low antigen dose (5 x 106 cells), NDTg/bcl-2 B cells resisted deletion induced by the Kk antigen (Fig. 1); however, this protection from death afforded by Bcl-2 overexpression was overcome with a 10-fold increased antigen dose (Fig. 1).
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mice bearing MT-Kb or KerIV-Kb transgenes, which target cell surface expression of the Kb protein to hepatocytes or epithelia, respectively (55, 56). Relative to antigen-free mice, antigen-bearing 3–83µ mice had profoundly reduced B cell numbers in the lymph nodes (Fig. 2 A, top; Fig. 2 B, compare H-2d to MT-Kb and KerIV-Kb), and substantial, but on average, incomplete deletion of the B cells in the spleen (Fig. 2 C, top; Fig. 2 D and reference 14). Control mice bearing antigen on all tissues (Fig. 2, H-2b) exhibited the phenotype of central B cell tolerance in which antigen-reactive cells were absent from the spleen. In the mice that demonstrated peripheral B cell deletion (3–83µ/MT-Kb or 3–83µ/KerIV-Kb), the remaining splenic cells manifested rapid turnover as assessed by their BrdU uptake over a 1-wk labeling period (Fig. 3). It is important to note that the MT-Kb/3–83µ mice showed a more profound tolerance than KerIV-Kb/3– 83µ mice, and had more complete elimination of Id+ B cells in the lymph nodes (Fig. 2) and of Id+ antibodies in the serum (Fig. 4 C).
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/MT-Kb and bcl-2/3–83µ/KerIV-Kb triple-Tg mice, enforced Bcl-2 expression partially or completely blocked peripheral B cell deletion to liver and skin antigens, respectively, and, on average, increased numbers of Id+ cells in both the lymph nodes and spleen (Fig. 2, A and C, bottom; Fig. 2, B and D). Interestingly, in the bcl-2/ 3–83µ/MT-Kb mice in which Bcl-2 protection was partial, the high rate of turnover of the autoreactive B cells was unchanged, whereas in the bcl-2/3–83µ/KerIV-Kb triple-Tg mice in which B cell deletion was effectively blocked, B cell turnover was also normalized, and only 20% of the B cells had incorporated BrdU over a 1-wk period (Fig. 3). Furthermore, lymph node cells from bcl-2/3–83µ/KerIV-Kb triple-Tg, but not those from bcl-2/3–83µ/MT-Kb mice, acquired the ability to respond to LPS stimulation (Fig. 4 A). The ability of lymph node and spleen cells to secrete 3–83 antibodies in vitro was consistent with the levels of 3–83 antibodies measured in the sera of these mice, as Id+ antibody levels were negligible in the bcl-2/3–83µ/MT-Kb mice, but were significant in the bcl-2/3–83µ/KerIV-Kb triple-Tg mice (Fig. 4 C). The only differences noted between bcl-2/3–83µ/KerIV-Kb triple-Tg mice and bcl-2/ 3–83µ ND controls were a consistent twofold reduction in surface IgM expression in the bcl-2/3–83µ/KerIV-Kb mice, suggesting antigen encounter that did not result in deletion (Fig. 2, A and C), and a lower percentage of cells expressing CD21 (data not shown). In both bcl-2/3–83µ/ KerIV-Kb and bcl-2/3–83µ/MT-Kb mice, the B220+ B cells coexpressed the maturation marker CD23 (data not shown). Collectively, these data demonstrated that Bcl-2 overexpression could inhibit peripheral B cell tolerance to both acute and chronic antigen exposure, but the degree of protection varied depending upon the dose or anatomical location of the antigenic stimulus.
Enforced Bcl-2 Expression Does Not Block Central B Cell Tolerance.
The Eµ–bcl-2-22 transgene drives expression of functionally active Bcl-2 in immature bone marrow B cells that normally lack Bcl-2 expression (46). To test the effect of this transgene on central B cell tolerance, we bred H-2b mice to 3–83µ and bcl-2/3–83µ mice, generating central deleting (CD)Tg and CDTg/bcl-2 mice, respectively. Like CDTg mice, CDTg/bcl-2 mice lacked Id+ B cells in the peripheral lymphoid organs (Fig. 2, H-2b; Table 1) and Id+ antibodies in the sera (Fig. 4 C). In addition, no Id+ antibodies were found in the supernatants of LPS-stimulated spleen cells from CDTg or CDTg/bcl-2 mice, whereas NDTg and NDTg/bcl-2 controls had significant levels of Id+ antibodies in both the sera and LPS culture supernatants (Fig. 4 A; data not shown). Since previous central tolerance studies investigating Bcl-2 overexpression used high-affinity antigens (KA 109 M–1), we tested whether or not Bcl-2 overexpression had perhaps a subtle, antigen affinity-dependent effect on tolerance induction by generating CDTg/bcl-2 mice expressing the Dk class I molecule to which 3–83 has very low affinity (KA 104 M–1; reference 58). Again the CDTg/ bcl-2 mice had no detectable increase of Id+ B cells in the peripheral lymphoid organs (Fig. 5) nor IgM idiotype in the serum (data not shown).
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lo, IgDlo, and CR1/2– (data not shown) which is indicative of immature B cells as has been previously reported in Bcl-2 overexpressing Ig Tg mice (9). Also indicative of immature B cells, these cells expressed RAG-2 messenger RNA transcripts (data not shown). These cells failed to secrete antibodies in LPS cultures and in vivo, as no antibodies were detected in the serum (Fig. 4 C). In CDTg/RAG-1–/– mice, no serum IgM was detected because RAG deficiency prevented development of Id– B cells (Fig. 4 D). These splenic immature B cells were short lived, as 50% of the cells had incorporated BrdU+ after 1 wk compared to only 20% of B220+ cells from non-Tg or NDTg mice (data not shown). Thus, in contrast to its effect on peripheral tolerance, the bcl-2 transgene appeared to have only a subtle effect on central B cell tolerance, allowing a subset of autoreactive nonfunctional, immature B cells to survive in the spleen for a short time.
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Tg mice by two- to fivefold (Fig. 7 B; Table 1). In contrast to the results with the mice bearing antigen targeted to peripheral tissues in which autoreactive B cells with enforced Bcl-2 expression were spared, the B cells appearing in the spleen and lymph nodes of CDTg/bcl-2 mice were nonautoreactive and had undergone receptor editing (Figs. 2 and 5). One clear indication of receptor editing was the appearance of B cells bearing both the Tg heavy chain, as detected with anti-IgDa, and endogenously encoded light chains, detected with 
chain–specific antibody (Fig. 7 A, and reference 49). The increase in the percentages of "edited" cells was the result of an increase in the total number of these cells in the lymphoid organs (Table 1). Thus, Bcl-2 overexpression apparently enhanced receptor editing or allowed survival of cells that had undergone receptor editing, or both.
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/ Ratio Suggests that Bcl-2 Overexpression Promotes Receptor Editing. and Ig- light chains would be the same in both CDTg and CDTg/bcl-2 mice. To test this notion, we measured the frequencies of Id– B cells that expressed or light chains. Fig. 8 A shows that most of the increase in peripheral B cells of the CDTg/bcl-2 mice could be accounted for by an increase in + B cells, suggesting that the bcl-2 transgene influences B cells undergoing light chain gene rearrangement. Interestingly, this elevated expression was also observed in bcl-2 Tg mice lacking Ig transgenes (Fig. 8 B), which consistently had a significantly higher percentage (17%) of + B cells compared to non-Tg mice (6% +).
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Consistent with the notion that tolerance mechanisms differ in immature and mature B cells, Bcl-2 overexpression was able to abrogate peripheral B cell tolerance, while only subtly altering central B cell tolerance. The ability of Bcl-2 to confer protection from deletion induced by peripherally expressed membrane self-antigen suggests that mature, autoreactive B cells are tolerized by apoptosis induction. These results are partly in agreement with the studies of Honjo's group (15, 46), who studied the effects of intraperitoneal injections of antiimmunoglobulins and self-erythrocytes, rather than foreign antigens, on peritoneal B cells that were largely of the B-1 lineage. The signals regulating apoptosis and immune tolerance in B-1 and B-2 subsets are likely to differ (5, 6, 18, 60). In our 3–83 Tg mice, the Id+ B cells, including those in the peritoneal cavity, are almost entirely B-2 B cells. Thus, our data are clear evidence for deletion of peritoneal B-2 B cells upon antigen encounter in vivo, which is inhibited by Bcl-2 overexpression in a dose-dependent manner. No such dose dependence was found in the study by Nisitani et al. (46), but analogous results have been obtained in bcl-2 transfected WEHI-231 cells in which anti-IgM–mediated death could be blocked at low but not high antibody dose (43). These differences may reflect a difference in the density of tolerogenic antigen expression, in BCR affinity for antigen, or in the relative tolerance susceptibility of B-1 and B-2 B cells.
Similar to the results with acute antigen administration, peripheral B cells tolerized by chronic exposure to natural autoantigen were variably rescued by enforced Bcl-2 overexpression. Furthermore, the extent of rescue afforded by the Bcl-2 transgene appeared to be inversely correlated with the degree of deletion in its absence: in MT-Kb mice, which normally show complete deletion of Id+ B cells from lymph nodes, enforced Bcl-2 only slightly inhibited deletion, whereas in the KerIV-Kb mice, which may express lower amounts of Kb in a relatively small subset of epithelial cells and in which autoreactive B cells are eliminated at a slower pace than in MT-Kb mice, B cell elimination was fully abrogated by Bcl-2. Because in this study the autoantigen and antibody receptor were kept constant, and only the nature and anatomical location of the antigenic stimulus were altered, the variable outcomes of Bcl-2 overexpression appear to be real effects, dependent on antigen density, concentration, in vivo location, or the developmental state of the B cell at time of antigen encounter. These results seem at odds with the paradigm developed from genetic studies in Caenorhabditis elegans that places the action of the nematode Bcl-2 homologue ced-9 at a distal point in the death signaling cascade (61). If this were also true in B cells, one might predict that Bcl-2 should protect from apoptosis over a wide range of antigen doses. It is therefore possible that the function of Bcl-2 is involved with the downstream integration of certain BCR signals. As Bcl-2 can titrate the death activity of heterodimerization partners, such as Bax (62–64), it is possible that the activity or quantity of these molecules can be affected by antigen signaling. It is interesting in this regard that Bcl-2 is subject to inactivation by inducible serine phosphorylation (65), a process that could conceivably be influenced by BCR signaling. It will be important to determine if higher doses of Bcl-2 can provide protection at higher antigen dose, or if cell death regulators distinct from Bcl-2 become limiting. It is interesting to note in this regard that in one study combined transgene expression of Bcl-2 and Bcl-xL had an additive, but still incomplete, effect in protection from anti-IgD–mediated B cell elimination (66). An alternative interpretation of our data is that strong tolerogenic signals through the BCR activate both Bcl-2–inhibitable and Bcl-2–resistant death pathways, whereas weaker tolerance signals activate only Bcl-2–inhibitable death pathways.
The observed weak or negligible effect of Bcl-2 overexpression on the rescue from the bone marrow of autoreactive B cells is in agreement with the study of Hartley et al. (9), who showed that Bcl-2 could delay turnover and permit limited peripheralization of immature autoreactive B cells, but could not promote their continued development to maturity. Young et al. have shown that Bcl-2 expression permits ectoptic accumulation of pre–B cells in the spleen (67). In RAG-sufficient mice, these peripheral, immature B cells are presumably capable of undergoing receptor editing even in response to antigens expressed exclusively in the periphery. This suggestion is supported by the upregulation of RAG-2 messenger RNA levels in spleens of CDTg/bcl-2/RAG-1–/– mice, which have a significant population of these immature B220+ cells (data not shown), and by the appearance of Id–, IgM+ B cells in spleens of 3–83/MT-Kb/bcl-2 mice (Fig. 1 C). These Id–, IgM+ cells express Tg heavy chain (data not shown) and are presumed to be the product of receptor editing. Collectively, these data show that Bcl-2 overexpression can manifest varying tolerance phenotypes depending on the maturational stage at which the B cell encounters autoantigen, promoting receptor editing in immature cells, and rescuing functional, mature cells from apoptosis.
How might Bcl-2 promote receptor editing? The ability of Bcl-2 to permit elevated steady-state numbers of immature and mature cells cannot alone explain the increased proportion of + B cells because increased numbers of cells undergoing editing, or surviving after editing, would not be expected to alter the Ig light chain / ratio. A more likely explanation is that the extended lifespan of autoreactive bone marrow B cells that is conferred by the bcl-2 transgene may provide an extended time window per cell for light chain gene rearrangement. The current models of murine light chain rearrangement suggest that rearrangements generally follow rearrangement by 24 h (68), and that most cells that turn over in the bone marrow do so before rearrangements are complete (69). This so-called crash-factor (70) suggests that mouse B cells normally have too little time to take full advantage of potential rearrangements that could rescue nonfunctional or autoreactive B cells. We propose that the bcl-2 transgene expression allows autoreactive B cells more potential rearrangement attempts, promoting the development of more nonautoreactive B cells bearing "edited" BCRs. This is consistent with data concerning transgene-driven Bcl-2 overexpression in thymocytes in which positive selection is enhanced as a result of increased endogenous TCR rearrangements, presumably due to the increased lifespan of the double-positive thymocytes expressing the bcl-2 transgene (1). The finding that Eu–bcl-2-22 Tg mice have consistently high percentages of + B cells suggests that receptor editing is enhanced even in the absence of antibody transgenes. Data from Rolink et al. have indicated that the Eµ–bcl-2-22 transgene prolongs in vitro survival of B cells undergoing light chain gene rearrangement resulting in high / ratios in cultured B cells (71).
One implication of our study is that altered or defective apoptosis in B cells encountering self-antigen in the periphery could provide a pool of potentially functional autoreactive B cells. In this study we have observed that the cells rescued from elimination by enforced Bcl-2 expression could sometimes also acquire functional reactivity. Further studies are needed to fully establish their functional capacity, but these results further suggest that although multiple independent levels of regulation are available to limit autoreactivity at each step in differentiation, tolerance to certain autoantigens may be definitively abrogated by a Bcl-2– inhibitable pathway.
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Acknowledgments |
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This work was supported by a Howard Hughes Medical Institute Predoctoral Fellowship (to J. Lang), by the Arthritis Foundation (to D. Nemazee), the National Institutes of Health (R01 GM 44809, R01 AI 33608, and K04 AI 01161; to D. Nemazee), and the National Health and Medical Research Council (Canberra, Australia; to A. Strasser). A. Strasser is a scholar of the Leukemia Society of America and a recipient of a Clinical Investigator Award from the Cancer Research Institute.
Submitted: 23 July 1997
Revised: 4 September 1997
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