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A Novel Benzodiazepine Increases the Sensitivity of B Cells to Receptor Stimulation with Synergistic Effects on Calcium Signaling and Apoptosis

2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês

10.1074/jbc.m403507200

1083-351X

Jeffrey J. Bednarski, Costas A. Lyssiotis, Rebecca Roush, Anthony E. Boitano, Gary D. Glick, Anthony W. Opipari,

Immune Cell Function and Interaction

Bz-423 is a 1,4-benzodiazepine with selective lymphotoxic properties and potent therapeutic activity against lupus-like disease in autoimmune mice. In NZB/W lupus-prone mice, Bz-423 specifically kills germinal center B cells, which are the cells that drive disease both in this model and in human systemic lupus erythematosus. In this report, the mechanistic basis for the selective action of Bz-423 is investigated. We show that Bz-423-induces superoxide as an immediate early response and that this reactive oxygen species is more effective as a second messenger death signal in B cells activated by B cell receptor stimulation compared with resting cells. As a result, low [Bz-423] that are not cytotoxic to non-stimulated cells kill stimulated cells in synergy with anti-immunoglobulin M antibodies. Subsequent experiments demonstrated that Bz-423 extends the rise in intracellular calcium that accompanies anti-immunoglobulin M stimulation, and this effect mediates the synergistic death response. Because B cell hyperactivation and altered calcium signaling is a distinguishing feature of autoreactive lymphocytes in lupus, the mechanism by which Bz-423 induces apoptosis preferentially targets disease-causing cells on the basis of their activation state. Thus, molecules like Bz-423 could form the basis for new and selective anti-lupus agents. Bz-423 is a 1,4-benzodiazepine with selective lymphotoxic properties and potent therapeutic activity against lupus-like disease in autoimmune mice. In NZB/W lupus-prone mice, Bz-423 specifically kills germinal center B cells, which are the cells that drive disease both in this model and in human systemic lupus erythematosus. In this report, the mechanistic basis for the selective action of Bz-423 is investigated. We show that Bz-423-induces superoxide as an immediate early response and that this reactive oxygen species is more effective as a second messenger death signal in B cells activated by B cell receptor stimulation compared with resting cells. As a result, low [Bz-423] that are not cytotoxic to non-stimulated cells kill stimulated cells in synergy with anti-immunoglobulin M antibodies. Subsequent experiments demonstrated that Bz-423 extends the rise in intracellular calcium that accompanies anti-immunoglobulin M stimulation, and this effect mediates the synergistic death response. Because B cell hyperactivation and altered calcium signaling is a distinguishing feature of autoreactive lymphocytes in lupus, the mechanism by which Bz-423 induces apoptosis preferentially targets disease-causing cells on the basis of their activation state. Thus, molecules like Bz-423 could form the basis for new and selective anti-lupus agents. Systemic lupus erythematosus (SLE) 1The abbreviations used are: SLE, systemic lupus erythematosus; GC, germinal center; NZB/W, (NZB x NZW)F1; BCR, B cell receptor; AICD, activation-induced cell death; MnTBAP, manganese(III)meso-tetrakis(4-benzoic acid)porphyrin; PI, propidium iodide; MPT, mitochondria permeability transition; ROS, reactive oxygen species; Z-VAD-fmk, benzyloxycarbonyl-VAD-fluoromethyl ketone; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraaceticacid; [Ca2+]i, intracellular calcium concentration; EC50, concentration of agent at which 50% of the cells are killed; ER, endoplasmic reticulum; InsP3, inositol-1,4,5-trisphosphate; O2·¯, superoxide; PH, peak height. is characterized by a spectrum of auto-antibodies that are the products of B cells that escape peripheral tolerance (1Diamond B. Katz J.B. Paul E. Aranow C. Lustgarten D. Scharff M.D. Annu. Rev. Immunol. 1992; 10: 731-757Crossref PubMed Scopus (223) Google Scholar). Although immunosuppressive, lymphotoxic drugs are effective for many patients, these drugs cause life-threatening side effects that account for a notable portion of lupus-related deaths (2Rubin L.A. Urowitz M.B. Gladman D.D. Q. J. Med. 1985; 55: 87-98PubMed Google Scholar). Therefore, agents with greater selectivity against disease-causing lymphocytes could significantly advance the treatment of SLE and related disorders. Because lymphocyte toxicity is an established treatment modality, it offers a starting point to develop new classes of therapeutic molecules. Toward this end, a library of 1,4-benzodiazepines was previously screened for cytotoxic members against Ramos B cells, a neoplastic B cell line with a germinal center (GC) phenotype (3Blatt N.B. Bednarski J.J. Warner R.E. Leonetti F. Johnson K.M. Boitano A. Yung R. Richardson B.C. Johnson K.J. Ellman J.A. Opipari Jr., A.W. Glick G.D. J. Clin. Invest. 2002; 110: 1123-1132Crossref PubMed Scopus (61) Google Scholar). These studies led to the identification of Bz-423 (Fig. 1), a pro-apoptotic molecule whose mechanism of action depends upon an increase in intracellular superoxide (O2·¯), produced as a result of the interaction of Bz-423 with a mitochondrial molecular target (3Blatt N.B. Bednarski J.J. Warner R.E. Leonetti F. Johnson K.M. Boitano A. Yung R. Richardson B.C. Johnson K.J. Ellman J.A. Opipari Jr., A.W. Glick G.D. J. Clin. Invest. 2002; 110: 1123-1132Crossref PubMed Scopus (61) Google Scholar). Comparison of Bz-423 with benzodiazepines used clinically and ligands of the peripheral benzodiazepine receptor reveals the unique cytotoxicity of this compound against B cells in vitro (3Blatt N.B. Bednarski J.J. Warner R.E. Leonetti F. Johnson K.M. Boitano A. Yung R. Richardson B.C. Johnson K.J. Ellman J.A. Opipari Jr., A.W. Glick G.D. J. Clin. Invest. 2002; 110: 1123-1132Crossref PubMed Scopus (61) Google Scholar). Based upon its lymphotoxic properties in vitro, we explored the cytotoxic properties of Bz-423 in two animal models of SLE, the MRL-lpr and the (NZB x NZW)F1 (NZB/W) mouse strains. Lupus-like disease in MRL-lpr mice is T-cell-dominated and is linked to defective Fas signaling. These defects allow a population of autoreactive CD4+ T cells to expand instead of undergoing apoptosis in response to physiologic cues (4Watanabe-Fukunaga R. Brannan C.I. Copeland N.G. Jenkins N.A. Nagata S. Nature. 1992; 356: 314-317Crossref PubMed Scopus (2737) Google Scholar, 5Watson M.L. Rao J.K. Gilkeson G.S. Ruiz P. Eicher E.M. Pisetsky D.S. Matsuzawa A. Rochelle J.M. Seldin M.F. J. Exp. Med. 1992; 176: 1645-1656Crossref PubMed Scopus (332) Google Scholar). In these animals, treatment with Bz-423 specifically reduced activated CD4+ cells (6Bednarski J.J. Warner R.E. Rao T. Leonetti F. Yung R. Richardson B.C. Johnson K.J. Ellman J.A. Opipari Jr., A.W. Glick G.D. Arthritis Rheum. 2003; 48: 757-766Crossref PubMed Scopus (39) Google Scholar). In contrast, an expanded population of activated B cells within GCs mediates disease in NZB/W mice. These activated B cells drive autoantibody production and pathogenicity that ultimately results in lupus nephritis (7Dixon F.J. Andrews B.S. Eisenberg R.A. McConahey P.J. Theofilopoulos A.N. Wilson C.B. Arthritis Rheum. 1978; 21: S64-S67Crossref PubMed Scopus (66) Google Scholar, 8Shlomchik M.J. Craft J.E. Mamula M.J. Nat. Rev. Immunol. 2001; 1: 147-153Crossref PubMed Scopus (473) Google Scholar). Administering Bz-423 to NZB/W mice selectively targeted activated GC B cells and reduced the number and size of GCs. These mice also had reduced autoantibody levels and improved glomerulonephritis. Although a complete molecular explanation for the abnormal GC persistence in NZB/W mice is not yet known, current evidence implicates defects in normal tolerance mechanisms, including defective B cell receptor (BCR)-mediated activation-induced cell death (AICD) (9Kozono Y. Kotzin B.L. Holers V.M. J. Immunol. 1996; 156: 4498-4503PubMed Google Scholar). In normal immune BALB/c mice, Bz-423 neither decreased viability nor increased apoptosis of splenic lymphocytes nor affected physiologic GC responses. Thus, at therapeutic doses, Bz-423 selectively kills disease-causing cells. Because GC-derived cells have also been shown to mediate disease pathogenesis in human SLE, we were particularly interested in understanding the mechanistic basis for the selectivity of Bz-423 in these cells. B cells acquire and maintain a GC phenotype through a process that depends upon stimulation of BCR. GC homeostasis also depends upon BCR-coupled apoptosis, which is defective in SLE and NZB/W mice (10Guzman-Rojas L. Sims-Mourtada J.C. Rangel R. Martinez-Valdez H. Immunology. 2002; 107: 167-175Crossref PubMed Scopus (62) Google Scholar). These observations suggested that BCR stimulation might be involved in enhancing sensitivity to Bz-423, resulting in the selectivity observed in the NZB/W studies. Therefore, we embarked on a line of investigation testing the hypothesis that BCR stimulation facilitates killing by Bz-423. Reagents—Bz-423 was synthesized as described previously (11Bunin B.A. Plunkett M.J. Ellman J.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 4708-4712Crossref PubMed Scopus (291) Google Scholar). Unless noted, all reagents were obtained from Sigma. Dihydroethidium (DHE), was obtained from Molecular Probes (Eugene, OR). Manganese(III)meso-tetrakis(4-benzoic acid)porphyrin (MnTBAP) was purchased from Alexis Biochemicals (San Diego, CA). Polyclonal goat anti-mouse immunoglobulin M (IgM) was obtained from ICN (Aurora, OH); soluble goat Fab2 anti-human IgM was obtained from Southern Biotechnology Associates (Birmingham, AL); anti-human CD40 monoclonal antibody clone 5C3 was obtained from PharMingen (San Diego, CA). Animals, Primary B Cells, Cell Lines, and Culture—6-week-old female Balb/C mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were euthanized and the spleens were removed for analysis. Splenocytes were obtained by mechanical disruption with isotonic lysis of red blood cells. B cell-rich fractions were prepared by negative selection using magnetic cell sorting with CD4, CD8a, and CD11b-coated microbeads (Miltenyi Biotec, Auburn, CA). Ramos cells were purchased from ATCC (Manassas, VA). Cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 μg/ml) and l-glutamine (290 μg/ml). Media for primary cells also contained 2-mercaptoethanol (50 μm). In vitro studies were performed in media containing 2% FBS and 0.5% Me2SO. Analysis of Lymphoid Cell Markers—Surface markers were detected with fluorescent-conjugated anti-Thy 1.2 (1 μg/ml, PharMingen) and/or anti-B220 (1 μg/ml, PharMingen). Samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences). B Cell Stimulation—Cell lines were stimulated with soluble goat Fab2 anti-human IgM (1 μg/ml) and/or purified anti-human CD40 (2.5 μg/ml). Primary mouse B cells were stimulated with affinity-purified goat anti-mouse IgM (20 μg/ml, ICN) immobilized in culture wells and/or soluble purified anti-mouse CD40 (clone HM40-3, 2.5 μg/ml, PharMingen). Bz-423 was added 10 min before stimuli were applied. Inhibitors were added 30 min prior to Bz-423. Determination of Synergy—Synergistic effects upon cytotoxicity between Bz-423 and anti-IgM were evaluated using isobologram analysis as described previously (12Gessner P.K. Toxicology. 1995; 105: 161-179Crossref PubMed Scopus (184) Google Scholar, 13Tallarida R.J. J. Pharmacol. Exp. Ther. 2001; 298: 865-872PubMed Google Scholar). For this analysis, two series of dosage curves were obtained. First, using fixed concentrations of Bz-423, the EC50 values (i.e. the concentration at which 50% of the cells are dead) of anti-IgM were measured. Then, at fixed concentrations of anti-IgM, the EC50 values of Bz-423 were obtained. These EC50 values were used to construct isobolograms. Detection of Cell Death and Intracellular ROS—Cell viability, hypodiploid DNA, and O2·¯ were measured by flow cytometry staining with propidium iodide (PI) and DHE, as described previously (3Blatt N.B. Bednarski J.J. Warner R.E. Leonetti F. Johnson K.M. Boitano A. Yung R. Richardson B.C. Johnson K.J. Ellman J.A. Opipari Jr., A.W. Glick G.D. J. Clin. Invest. 2002; 110: 1123-1132Crossref PubMed Scopus (61) Google Scholar). Measurement of Intracellular Calcium Concentration [Ca2+]i Using Fura 2—The cell-permeable acetoxymethyl ester form of Fura 2 (2.5 μm, Fura 2-AM) was added to Ramos cells (107/ml) in loading buffer (1 mm CaCl2, 1 mm MgCl2, 1% FBS). Fura 2-AM is retained in the cell after being de-esterified by esterases in the cytoplasm; when excited at two alternate wavelengths (340 and 380 nm), the ratio of fluorescence emission at 510 nm is related to [Ca2+]i independently of intracellular dye concentration. After incubating 30 min at 37 °C to load with Fura 2, cells were washed and then resuspended in loading buffer at 106/ml; 200 μl (2 × 105 cells) were added per well to a 96-well plate. As indicated, cells were pre-incubated with Bz-423 or solvent control for 2 min, warmed to 37 °C, and anti-IgM (1 μg/ml) was added, immediately after which fluorescence (510 nm) was monitored every 15 s over 8 min using a microplate spectrofluorometer (Molecular Devices). Fluorescence specific to Fura 2 was calculated as the difference between the fluorescence intensities from Fura 2-loaded cells and unloaded cells. The maximal fluorescence at 380 nm determined by the addition of ionophore Br-A23187 (2 μg/ml) and minimum fluorescence at 340 nm determined by the addition of EGTA (35 mm) to Br-A23187-treated cells were used to calculate [Ca2+]i as described (14Hirst R.A. Harrison C. Hirota K. Lambert D.G. Methods Mol. Biol. 1999; 114: 31-39PubMed Google Scholar). Detection of [Ca2+]i within Single Cells using Flow Cytometry—Ramos cells (107/ml) were incubated (30 min, 37 °C) with the cell-permeable fluorescent dye Fluo-3 AM (4 μg/ml) in loading buffer (1 mm CaCl2, 1 mm MgCl2, 1% FBS) containing Pluronic (0.02%) and Probe-necid (4 μm). The cells were washed with phosphate-buffered saline and resuspended in loading buffer at 106/ml. Cell suspensions were incubated with Bz-423 for 10 min at room temperature, warmed to 37 °C, then analyzed on the flow cytometer continuously for 10 min with the temperature maintained at 37 °C. [Ca2+]i was calculated as described previously based upon maximum Fluo-3 fluorescence (determined by the addition of 2 μg/ml Br-A23187) and minimum fluorescence (determined by addition of 35 mm EGTA to Br-A23187-treated cells). Statistical Analysis—Statistical analysis was conducted by using the SPSS software package. All data are presented as mean ± S.D. BCR-stimulation Sensitizes Cells to Bz-423—B cells acquire and maintain a GC phenotype through a process that depends upon stimulation of BCRs (10Guzman-Rojas L. Sims-Mourtada J.C. Rangel R. Martinez-Valdez H. Immunology. 2002; 107: 167-175Crossref PubMed Scopus (62) Google Scholar). To account for the selective GC reduction in the treated NZB/W mice, we postulated that BCR stimulation facilitates killing by Bz-423. To test this hypothesis, primary B cells were isolated by negative selection from splenocytes harvested from Balb/C mice. B cell-enriched isolates (>95% B220+/Thy1.2-) were incubated with immobilized polyclonal anti-IgM to extensively cross-link BCRs. This strong stimulus expectedly provoked AICD in ∼40% of cells (Fig. 2, white bars). When added alone, Bz-423 (4 μm) killed 20% of the cells (black bars). When Bz-423 (4 μm) was combined with anti-IgM, killing was greater than with either agent alone (gray bars). Cells were also co-stimulated during these treatments with antibody specific for CD40, because CD40 stimulation is a GC cell-survival signal linked with lupus pathogenesis (15Choi Y.S. Immunol. Res. 1997; 16: 161-174Crossref PubMed Scopus (36) Google Scholar, 16Cheng G. Schoenberger S.P. Curr. Dir. Autoimmun. 2002; 5: 51-61Crossref PubMed Google Scholar). In the presence of anti-CD40, killing by anti-IgM alone was completely abrogated (40% killing decreased to 5%; see Fig. 2) and the response to Bz-423 was also decreased (20 to 9%.) Interestingly, however, the response to Bz-423 plus anti-IgM was not reduced to the same extent by CD40 (anti-IgM alone, 5%; Bz-423 alone, 9%; anti-IgM plus Bz-423, 38% killing). These data indicate that in primary B cells given a survival stimulus through CD40, BCR and Bz-423 cooperate to increase cytotoxic effects. Because primary B cells can not be maintained in culture over long periods of time, we chose to further investigate synergy in the immortalized, follicular B cell lymphoma Ramos cell line. Ramos cells are an Epstein-Barr virus-negative B cell lymphoma line and express surface markers and Bcl-6 consistently with a GC phenotype (17Klein G. Giovanella B. Westman A. Stehlin J.S. Mumford D. Intervirology. 1975; 5: 319-334Crossref PubMed Scopus (345) Google Scholar, 18Moriyama M. Yamochi T. Semba K. Akiyama T. Mori S. Oncogene. 1997; 14: 2465-2474Crossref PubMed Scopus (36) Google Scholar). More importantly, because Ramos cells mount an apoptotic response to BCR ligation like GC cells (19Kim D. Hur D.Y. Kim Y.S. Lee K. Lee Y. Cho D. Kang J.S. Kim Y.I. Hahm E. Yang Y. Yoon S. Kim S. Lee W.B. Park H.Y. Kim Y.B. Hwang Y.I. Chang K.Y. Lee W.J. Hum. Immunol. 2002; 63: 576-587Crossref PubMed Scopus (7) Google Scholar, 20Cragg M.S. Zhang L. French R.R. Glennie M.J. Br. J. Cancer. 1999; 79: 850-857Crossref PubMed Scopus (20) Google Scholar), this line was suitable to examine the synergy between Bz-423 and BCR stimulation on cell death. Soluble anti-IgM Fab2 dose-dependently killed Ramos cells (Fig. 3A). When used at limiting concentrations ( 8-fold over baseline, compared with 10% of control cells, and the elevation is prolonged. After 10 min, 64% of cells continued to have a fluorescent intensity above the baseline (Fig. 6). In the control cells, a rapid increase in calcium was induced by Fab2 and resolved within 3 min, after which time, > 90% of cells had fluorescence at or below the median fluorescence intensity of unstimulated cells. Thus, by contrast, the calcium response in cells treated with Bz-423 is both amplified and prolonged. BAPTA, which chelates extracellular calcium and blocks synergistic killing, was used in these experiments to determine whether the increased [Ca2+]i resulting from Bz-423 depends upon extracellular Ca2+. Fluo-3-loaded cells were pre-incubated (30 min) with BAPTA prior to Bz-423 or vehicle control. When cells pre-treated with either BAPTA were treated with Bz-423 and then anti-IgM Fab2, the increase in [Ca2+]i associated with Bz-423 was significantly blunted (Fig. 6). As well, BAPTA reduced the calcium response to anti-IgM Fab2 in the absence of Bz-423. Because vitamin E reduces Bz-423-induced O2·¯, it was used to determine the functional importance of ROS in modulating [Ca2+]i. Vitamin E also significantly reversed the effects that Bz-423 had upon anti-IgM-induced [Ca2+]i (Fig. 6). In contrast, vitamin E had no effect upon the calcium response to anti-IgM in the absence of Bz-423. Taken together, our data show that Bz-423 dramatically increases BCR-coupled calcium signaling and that Bz-423-induced ROS is critical for affecting the increase. Thus, Bz-423 plus anti-IgM produce a synergistic death response because their individual signaling pathways intersect to coordinately affect [Ca2+]i. The selectivity of Bz-423 against GC B cells in autoimmune NZB/W mice prompted these studies, which have identified synergy between the cytotoxic effects of receptor stimulation and Bz-423 in B cells. Functionally, this synergy means that B cells undergo significant apoptosis in response to amounts of Bz-423 and anti-IgM that, as single agents, are not cytotoxic. Although ROS are not necessary for BCR-coupled AICD, the O2·¯ generated by Bz-423 is critical for the synergistic response. Given that O2·¯ does not diffuse across membranes (24Gus'kova R.A. Ivanov I.I. Kol'tover V.K. Akhobadze V.V. Rubin A.B. Biochim. Biophys. Acta. 1984; 778: 579-585Crossref PubMed Scopus (93) Google Scholar) and only a small fraction of superoxide is vectorially released into the intermembrane space and enters the cytosol through voltage-dependent anion channels in the outer membrane (25Han D. Williams E. Cadenas E. Biochem. J. 2001; 353: 411-416Crossref PubMed Scopus (488) Google Scholar, 26Han D. Antunes F. Canali R. Rettori D. Cadenas E. J. Biol. Chem. 2003; 278: 5557-5563Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar), how does O2·¯ induced in mitochondria by Bz-423 affect BCR-coupled calcium responses that are regulated by cytosolic enzymes and other organelles? O2·¯ generated within the mitochondrial matrix is converted to H2O2 by manganese superoxide dismutase in the matrix. H2O2 freely and rapidly diffuses through membranes and enters the cytosol (27Antunes F. Cadenas E. FEBS Lett. 2000; 475: 121-126Crossref PubMed Scopus (435) Google Scholar). In the reducing intracellular environment, O2·¯, H2O2, and/or -OH (the product of H2O2 breakdown in the Fenton reaction) are able to oxidize a variety of protein and lipid moieties throughout the cell, which ultimately results in the modulation of Ca2+ signaling. Hence, it is likely that H2O2 is the ROS that mediates the observed effects. Our findings show that the synergistic response depends upon extracellular calcium. However, because calcium influx across the plasma membrane is coupled to depletion of intra-cellular calcium stores in B cells (28Kiselyov K. Shin D.M. Shcheynikov N. Kurosaki T. Muallem S. Biochem. J. 2001; 360: 17-22Crossref PubMed Scopus (56) Google Scholar), the present data do not necessarily indicate that Bz-423-induced ROS directly affects calcium channels or pumps in the plasma membrane. Although this is a possibility, extracellular calcium sources may be engaged more indirectly, through capacitative regulation. There are multiple mechanisms by which Bz-423-induced ROS might modulate Ca2+ signaling. One possibility is that Bz-423-induced ROS ultimately increases signals in the BCR-induced cell death pathway that result in sustained Ca2+ levels. Bruton's tyrosine kinase and Syk (two of several protein tyrosine kinases activated by the BCR; Ref. 29Kurosaki T. Maeda A. Ishiai M. Hashimoto A. Inabe K. Takata M. Immunol. Rev. 2000; 176: 19-29Crossref PubMed Scopus (138) Google Scholar) are candidates for points of intersection, because H2O2 induces their activation (30Qin S. Stadtman E.R. Chock P.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7118-7123Crossref PubMed Scopus (50) Google Scholar, 31Takano T. Sada K. Yamamura H. Antioxid. Redox. Signal. 2002; 4: 533-541Crossref PubMed Scopus (44) Google Scholar) and each has a critical role in determining calcium signaling by phosphorylating phospholipase Cγ (32Takata M. Kurosaki T. J. Exp. Med. 1996; 184: 31-40Crossref PubMed Scopus (426) Google Scholar). The BCR initially phosphorylates and activates Syk as well as phosphatidylinositol 3-kinase. Phosphatidylinositol 3-kinase generates the second messenger phosphatidylinositol 3,4,5-trisphosphate that mediates membrane recruitment of Bruton's tyrosine kinase which, along with Syk, phosphorylates and activates phospholipase Cγ (29Kurosaki T. Maeda A. Ishiai M. Hashimoto A. Inabe K. Takata M. Immunol. Rev. 2000; 176: 19-29Crossref PubMed Scopus (138) Google Scholar). Inositol-1,4,5-trisphosphate (InsP3) generated by phosphorylating phospholipase Cγ raises [Ca2+]i by binding to InsP3 receptors in the endoplasmic reticulum (ER) membrane, depleting stores within the ER and triggering influx through the plasma membrane (33Bijsterbosch M.K. Rigley K.P. Klaus G.G. Biochem. Biophys. Res. Commun. 1986; 137: 500-506Crossref PubMed Scopus (78) Google Scholar). Bruton's tyrosine kinase is a particularly appealing candidate, because when Bruton's tyrosine kinase is persistently activated, for example, by point mutation in its pleckstrinhomology domain, BCR cross-linking generates a sustained increase in [Ca2+]i identical to the response observed in our experiments (34Fluckiger A.C. Li Z. Kato R.M. Wahl M.I. Ochs H.D. Longnecker R. Kinet J.P. Witte O.N. Scharenberg A.M. Rawlings D.J. EMBO J. 1998; 17: 1973-1985Crossref PubMed Scopus (358) Google Scholar). Alternatively, because the ER lies close to the mitochondria, ROS emanating from the mitochondria might directly regulate Ca2+ release channels in the ER (ryanodine receptors and InsP3 receptors) or alter mechanisms that normally lower [Ca2+]i such as re-uptake by the Ca2+-ATPase in the ER (35Kourie J.I. Am. J. Physiol. 1998; 275: C1-C24Crossref PubMed Google Scholar). Calcium-specific channels and pumps are sensitive to modulation by ROS either through direct oxidation of sulfhydryl groups located on the ion transport proteins (36Cai S. Sauve R. J. Membr. Biol. 1997; 158: 147-158Crossref PubMed Scopus (53) Google Scholar, 37Jeulin C. Dazy A.C. Marano F. Pflügers Arch. Eur. J. Physiol. 2002; 443: 574-583Crossref PubMed Scopus (13) Google Scholar), peroxidation of membrane phospholipids (38Burton K.P. Morris A.C. Massey K.D. Buja L.M. Hagler H.K. J. Mol. Cell Cardiol. 1990; 22: 1035-1047Abstract Full Text PDF PubMed Scopus (119) Google Scholar), or inhibition by lower ATP levels (39DiPolo R. Beauge L. Prog. Biophys. Mol. Biol. 2002; 80: 43-67Crossref PubMed Scopus (14) Google Scholar). Bz-423-induced ROS could also modulate ER channels in a more indirect manner, using cytochrome c as a messenger to the ER. A recent description (40Boehning D. Patterson R.L. Sedaghat L. Glebova N.O. Kurosaki T. Snyder S.H. Nat. Cell Biol. 2003; 5: 1051-1061Crossref PubMed Scopus (541) Google Scholar) of the interplay between calcium and cytochrome c coordinating mitochondrial-ER interactions and driving apoptosis provides another explanation for the exaggerated calcium response and insight into how increased calcium may trigger apoptosis. Boehning et al. (40Boehning D. Patterson R.L. Sedaghat L. Glebova N.O. Kurosaki T. Snyder S.H. Nat. Cell Biol. 2003; 5: 1051-1061Crossref PubMed Scopus (541) Google Scholar) identified small-scale release of cytochrome c from mitochondria in response to agents including staurosporine and ceramide. Because Bz-423 acts directly on mitochondria, it is conceivable that it also causes a similar cytochrome c release. Boehning et al. then showed that the small amount of cytochrome c released is sufficient to bind to and promote calcium conductance through InsP3 receptors in the ER. The released calcium feeds back to the mitochondria, triggering a coordinate permeability transition and massive cytochrome c release from all mitochondria in the cell, ultimately engaging the caspase-dependent death machinery. It is possible that in an analogous fashion, the direct actions of Bz-423 on the mitochondria lead to a similar early, small-scale cytochrome c response that contributes to the increased BCR-induced calcium release by sensitizing InsP3 receptors. Because [Ca2+]i is fundamental for B cell activation, proliferation, and apoptosis, it is not surprising that the changes in calcium signaling effected by Bz-423 are functionally important. Among the parameters that influence BCR-driven apoptosis, the magnitude of the rise in [Ca2+]i induced by receptor ligands is directly correlated with the apoptotic outcome induced by BCR stimulation (41Grafton G. Goodall M. Gregory C.D. Gordon J. Cell. Immunol. 1997; 182: 45-56Crossref PubMed Scopus (17) Google Scholar). Furthermore, the magnitude and duration of the intracellular calcium response to BCR ligation precisely determines the differential triggering of the transcriptional regulators nuclear factor-κB, c-Jun NH2-terminal kinase, nuclear factor of activated T cells, and extracellular signal-regulated kinase that ultimately dictate the characteristics of a cellular response (42Dolmetsch R.E. Lewis R.S. Goodnow C.C. Healy J.I. Nature. 1997; 386: 855-858Crossref PubMed Scopus (1563) Google Scholar). Thus, in addition to influencing BCR-coupled calcium signals in AICD, Bz-423 may modulate other B cell responses to antigen receptor stimulation. In summary, we have demonstrated that the cytotoxic, immunomodulatory agent Bz-423 synergizes with BCR stimulation, and it is likely that this effect accounts (at least in part) for the selectivity observed in vivo. Considering that other receptors on B cells, which are effectively targeted by therapeutic antibodies presently in clinical use, are also coupled to calcium signals (for example the CD20 B cell-specific surface molecule targeted by Rituxan; Ref. 43Chinn P. Braslawsky G. White C. Hanna N. Cancer Immunol. Immunother. 2003; 52: 257-280Crossref PubMed Scopus (45) Google Scholar), it may be possible to exploit Bz-423 for cancer chemotherapy. Our findings suggest how the properties of Bz-423 could provide therapeutic utility for autoimmunity, and current efforts are aimed at evaluating these possibilities.