Fas-induced B Cell Apoptosis Requires an Increase in Free Cytosolic Magnesium as an Early Event*

Millie M. ChienDagger , K. Elizabeth ZahradkaDagger §, M. Karen Newell, and John H. FreedDagger §parallel

From the Dagger  Division of Basic Immunology, Department of Medicine, National Jewish Medical and Research Center, Denver, Colorado 80206 and the § Department of Immunology, University of Colorado Health Sciences Center, Denver, Colorado 80262

    ABSTRACT
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Abstract
Introduction
References

Ligation of the Fas molecule expressed on the surface of a cell initiates multiple signaling pathways that result in the apoptotic death of that cell. We have examined Mg2+ mobilization as well as Ca2+ mobilization in B cells undergoing Fas-initiated apoptosis. Our results indicate that cytosolic levels of free (non-complexed) Mg2+ ([Mg2+]i) and Ca2+ ([Ca2+]i) increase in cells undergoing apoptosis. Furthermore, the percentages of cells mobilizing Mg2+, fragmenting DNA, or externalizing phosphatidylserine (PS) increase in parallel as the concentration of anti-Fas monoclonal antibody is raised. Kinetic analysis suggests that Mg2+ mobilization is an early event in apoptosis, clearly preceding DNA fragmentation and probably occurring prior to externalization of PS as well. The source of Mg2+ that produces the increases in [Mg2+]i is intracellular and most likely is the mitochondria. Extended pretreatment of B cells with carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial oxidative phosphorylation, produces proportional decreases in the percentage of cells mobilizing Mg2+, fragmenting DNA, and externalizing PS in response to anti-Fas monoclonal antibody treatment. These observations are consistent with the hypothesis that elevated [Mg2+]i is required for apoptosis. Furthermore, we propose that the increases in [Mg2+]i function not only as cofactors for Mg2+-dependent endonucleases, but also to facilitate the release of cytochrome c from the mitochondria, which drives many of the post-mitochondrial, caspase-mediated events in apoptotic cells.

    INTRODUCTION
Top
Abstract
Introduction
References

Signal transduction by cell-surface receptors is critical for the regulation of cell growth and differentiation, but may serve a variety of other functions, including induction of programmed cell death or apoptosis. The cell-surface receptor Fas (CD95), which belongs to the tumor necrosis factor/nerve growth factor receptor family, is the most extensively studied of the so-called death receptors (1, 2). Cross-linking Fas molecules on the cell surface either by Fas ligand or by anti-Fas monoclonal antibody (mAb)1 results in the apoptotic death of the cell. There have been a number of studies of the phenotypic changes that occur early in apoptosis. Among those events, the disruption of the mitochondrial transmembrane potential (Delta psi m) (3, 4) and the aberrant exposure of phosphatidylserine (PS) have been studied extensively (5, 6). It has also been reported that a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) can trigger lethal processes (7) and that increased intracellular Mg2+ concentration ([Mg2+]i) promotes apoptosis in rat hepatocytes (8). Most recently, Eskes et al. (9) demonstrated that the release of cytochrome c from mitochondria (induced by overexpression of the apoptosis-promoting molecule Bax) is independent of Delta psi m, but dependent on Mg2+. Although Mg2+ has often been considered a messenger in signal transduction because of its ability to regulate a variety of cellular processes, changes in [Mg2+]i following induction of apoptosis via receptor ligation in lymphocytes have not been demonstrated.

The susceptibility of primary B cells to Fas-mediated apoptosis is dependent on whether the cell has received signals in the absence of antigen through CD40, another member of the tumor necrosis factor receptor superfamily. Resting B cells express very low levels of Fas and are not prone to apoptosis induced by this receptor (10, 11). Previous investigators (12-15) have shown that engagement of CD40 can induce high levels of Fas expression and Fas sensitivity in primary B cells. In this study, we have used Fas-positive, CD40-primed primary B cells or the M12.C3 B cell lymphoma, which also expresses high levels of Fas, to explore the role of [Mg2+]i in Fas-induced apoptosis. Our results demonstrate that B cells undergoing apoptosis have elevated [Mg2+]i and that this increase in free cytosolic Mg2+ appears to serve as a "second messenger" for downstream events in apoptosis.

    EXPERIMENTAL PROCEDURES

Materials-- Chemicals were purchased from Sigma unless stated otherwise. Mag-indo-1 AM and indo-1 AM were purchased from Molecular Probes, Inc. (Eugene, OR). The ApoAlert annexin V apoptosis kit was obtained from CLONTECH (Palo Alto, CA). Baby rabbit complement and Lympholyte-M were purchased from Accurate Chemical & Scientific Corp. (Westbury, NY). Fetal bovine serum was purchased from Life Technologies, Inc. Ionomycin was obtained from Calbiochem. Anti-Fas mAb (Jo2) was purchased from Pharmingen (San Diego, CA). The hybridoma secreting the anti-CD40 mAb 1C10 was a gift from Maureen Howard (16). Hybridomas secreting anti-CD4 (GK 1.5) and anti-CD8 (HO = 2.2) antibodies were obtained from American Type Culture Collection. The hybridoma secreting anti-Thy 1.2 antibody (T24) was obtained from Dr. Uwe Staerz (National Jewish Medical and Research Center). All mAbs were purified from culture supernatants by passage over protein A-Sepharose (Sigma). RPMI 1640 complete medium (Cellgro, Herndon, VA) was supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 40 µg/ml gentamicin, 100 units/ml penicillin G, 100 µg/ml streptomycin, and 50 µM 2-mercaptoethanol. Hanks' balanced salt solution (HBSS) and modified HBSS lacking Mg2+ and Ca2+ were obtained from Sigma.

Cells-- AKR/J mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and were used at 6-10 weeks of age. Splenic B cells were prepared from murine splenocytes after the depletion of T cells by a mixture of anti-CD4 (GK 1.5), anti-CD8 (HO = 2.2), and anti-Thy 1.2 (T24) mAbs with baby rabbit complement as described previously (17). The isolated primary B cells (5 × 106/ml) were incubated with anti-CD40 antibody (1C10) at 8 µg/ml of RPMI 1640 complete medium in six-well tissue culture plates (5.5 ml/well) at 37 °C and 5% CO2 for 3 days (13). The CD40-stimulated B cells were washed with phosphate-buffered saline, and the viable cells were obtained by sedimentation of dead cells with Lympholyte-M. The isolated activated B cells had high Fas expression as determined by staining with fluorescein isothiocyanate-labeled anti-Fas mAb Jo2. The cell line used in this study is the B cell lymphoma M12.C3, which is maintained in RPMI 1640 complete medium and also has high Fas expression. Both types of cells were treated with varying concentrations of anti-Fas mAb in RPMI 1640 complete medium at 37 °C and 5% CO2.

Determination of Mg2+ and Ca2+ Mobilization-- Untreated and anti-Fas mAb-treated cells were washed with HBSS, loaded with 20 µM mag-indo-1 AM or indo-1 AM in HBSS (pH 7.0) containing 10 mM HEPES for 30 min at 37 °C, diluted 2-fold with prewarmed HBSS (pH 7.4) containing 5% fetal bovine serum and 10 mM HEPES; and incubated for an additional 30 min as described previously (18). The fluorochrome-loaded cells were then washed and resuspended in HBSS (pH 7.2) containing 5% fetal bovine serum and 10 mM HEPES for flow cytometric analysis. In the experiments in which the role of extracellular Ca2+ and Mg2+ was explored, the cells were washed, resuspended, and analyzed in modified HBSS containing only the appropriate divalent cation(s). An Ortho 50H cytometer with a 5-watt argon laser set for 364 nm excitation at 50 milliwatts was used. Fluorescence emission data were collected through a 390/490-nm band-pass filter using Phoenix Flow Acquisition software. The ratio of emissions for 390/490 nm was determined, and a cytogram of [Mg2+]i or [Ca2+]i was constructed by reference to standard curves of 390/490 nm emission ratio versus cation concentration (19). Readily mobilized pools of Mg2+ and Ca2+ in the cells (106/ml/sample) were measured by treating the mag-indo-1- and indo-1-loaded cells with 1 µM ionomycin. Flow cytometric analysis was begun before addition of ionomycin to obtain base-line values. The cell flow was halted for addition of ionomycin, resulting in a gap in the profiles. The data were analyzed using Phoenix Flow System MTIMENEW software, and mean [Mg2+]i and [Ca2+]i were calculated as functions of time. In addition, the percentage of cells mobilizing Mg2+ after ionomycin treatment was determined for each time point, and maximum values were calculated.

Phosphatidylserine Expression-- PS expression was determined using an ApoAlert annexin V apoptosis kit. The untreated and anti-Fas mAb-treated cells were washed with phosphate-buffered saline, and the washed cells (5 × 105) were resuspended in 200 µl of 1× binding buffer. After adding 5 µl each of fluorescein-labeled annexin V (0.5 µg/ml final concentration) and propidium iodide (PI; 1 µg/ml final concentration), the treated cells were incubated at room temperature for 15 min in the dark; the cells were then analyzed on a FACSCalibur flow cytometer (Becton Dickinson Advanced Cellular Biology, San Jose, CA). Two-color analysis allowed the resolution of viable (PS-PI-), early apoptotic (PS+PI-), and late apoptotic and necrotic (PS+PI+) populations.

DNA Fragmentation-- Cells (5 × 105) were incubated in 200 µl of a solution containing PI (5 µg/ml), saponin (0.3%), RNase (50 µg/ml), and EDTA (5 mM) for 30 min at room temperature (20). Apoptotic cells were identified on a FACSCalibur flow cytometer as a distinct hypodiploid population that showed a diminished staining relative to the G0/G1 population of the normal viable cells.

    RESULTS

Fas-mediated Mg2+ and Ca2+ Mobilization in B Cell Lymphoma Cells-- To study the effect of Fas ligation on [Mg2+]i and [Ca2+]i, we conducted a flow cytometric analysis of M12.C3 B cell lymphomas that had been incubated with anti-Fas mAb and then loaded with the Mg2+ and Ca2+ indicators mag-indo-1 and indo-1, respectively. Untreated cells appeared as a homogeneous population by forward side scatter (data not shown) with uniform base-line distributions of [Mg2+]i and [Ca2+]i (Fig. 1, A and B, respectively; 0~2 min). The mean value of [Mg2+]i in the untreated cells was 500 µM (Fig. 1E, dashed line at 0~2 min), in good agreement with the range of values reported for hepatocytes (21), vascular smooth muscle cells (22), and cardiac muscle cells (23). The mean value for [Ca2+]i was 100 nM (Fig. 1F, dashed line at 0~2 min), in agreement with the published value of 90 nM for [Ca2+]i in murine splenic B cells (19). Incubating M12.C3 cells for 3 h with anti-Fas mAb (0.5 µg/ml) produced two distinguishable subpopulations with regard to [Mg2+]i. In the anti-Fas mAb-treated cells, one population had a base-line [Mg2+]i of 500 µM (Fig. 1C, arrowheads), and a second population had a base-line [Mg2+]i of 900 µM (Fig. 1C, 0~2 min). The existence of these two populations is reflected in the mean base-line [Mg2+]i of 700 µM for the treated cells (Fig. 1E, solid line, 0~2 min). Intracellular Ca2+ was monitored in parallel by flow cytometric analysis of indo-1-loaded cells. Fas cross-linking also gave rise to two separate populations, a well defined major one with a [Ca2+]i of 100 nM and a broad "tail" with Ca2+i levels ranging up to 1000 nM or higher (Fig. 1D, 0~2 min). These populations produced an increase in the mean base-line [Ca2+]i from 100 nM in the untreated cells to 170 nM in the anti-Fas mAb-treated cells (Fig. 1F, solid line, 0~2 min).


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Fig. 1.   Fas-mediated Mg2+ and Ca2+ mobilization in B cell lymphoma cells. M12.C3 cells (106/ml) were incubated with 0.5 µg/ml anti-Fas mAb for 3 h at 37 °C, and then untreated control and Fas-treated cells were loaded with mag-indo-1/AM or indo-1/AM. Shown is an isometric display of [Mg2+]i and [Ca2+]i of the control (A and B) and anti-Fas (0.5 µg/ml)-treated (C and D) M12.C3 cells. Once base-line levels of [Mg2+]i and [Ca2+]i were established, 1 µM ionomycin was added at the time indicated by the arrows, and [Mg2+]i and [Ca2+]i were monitored for an additional 8 min. Mean cation concentrations were calculated from the isometric data (E and F). The dip indicates the addition of ionomycin to the flow cells. Values before the dip are base-line values, and differences between the treated and control samples indicate cation mobilization induced by anti-Fas mAb treatment alone. Values after the dip indicate the extent of further cation mobilization induced by ionomycin. The solid lines show the response of cells treated with anti-Fas mAb, and the dashed lines show that of the control cells. The percentage of cells mobilizing Mg2+ in response to treatment with ionomycin was calculated from the isometric data (G). Ionomycin was added at the times indicated in E. The solid line shows the response of cells after treatment with anti-Fas mAb, and the dashed line shows that of the untreated control cells. Cells piling up on the left-hand axis in B-D are not shown for clarity. They are, however, included in the calculations for E-G.

After the base-line values for [Mg2+]i and [Ca2+]i were established, the cells were treated in the flow cytometer with 1 µM ionomycin and were monitored for several minutes longer (~2-10 min) to determine if there are pool(s) of divalent cations that can be mobilized by ionophore treatment. In the untreated cells, ionomycin produced the classical Ca2+ mobilization pattern with an initial sharp rise followed by a gradual decrease to a plateau value somewhat above base-line levels (Fig. 1, B and F, dashed line). Untreated cells did not have a pool of Mg2+ that could be mobilized by this same dose of ionomycin and showed an insignificant increase in the mean [Mg2+]i (Fig. 1E, dashed line) as well as a transient response by no more than 22% of the cells in the population (Fig. 1G, dashed line). Ionomycin treatment of cells that had been incubated with anti-Fas mAb revealed significant differences from the control cells. The vast majority of the high [Mg2+]i cells clearly had a pool of Mg2+ that could be further mobilized by ionomycin treatment (Fig. 1, C and G, solid line), which is reflected in the gradual rise in the mean [Mg2+]i, reaching maximum values by 8-10 min after ionomycin treatment (Fig. 1E, solid line). The low [Mg2+]i population (Fig. 1C, arrowheads) did not further mobilize Mg2+ in response to ionomycin. Ionomycin treatment also revealed that the cells that had been incubated with anti-Fas mAb fell into two populations with regard to Ca2+ mobilization. Although both subpopulations responded to ionomycin treatment, one continued to show the "classic" mobilization profile of the untreated cells, whereas the other reached and maintained extremely high Ca2+i levels (Fig. 1D). The behavior of these populations is reflected in the increased mean [Ca2+]i response (Fig. 1F, solid line). Because both subpopulations mobilized Ca2+, it was not possible to use the flow cytometer software to calculate a percent response for Ca2+ in order to discriminate between them.

The Percentage of Cells in the High [Mg2+]i State, Which Further Mobilize Mg2+ upon Ionomycin Treatment, Correlates with the Percentage of Cells That Die by Apoptosis-- Treatment of M12.C3 cells with increasing concentrations of anti-Fas mAb for 16 h caused a dose-dependent increase in the percentage of cells in the high [Mg2+]i state (Fig. 2A). This is also reflected in a dose-dependent increase in the mean [Mg2+]i, which reached a plateau value of 700 µM with 0.03 µg/ml anti-Fas mAb (Fig. 2C, closed squares). Similarly, the percentage of cells in the high [Ca2+]i state increased in a dose-dependent manner (Fig. 2B), and the base-line [Ca2+]i increased from 100 to 170 nM (Fig. 2D, closed circles). These latter observations are consistent with the generally accepted model that elevation of [Ca2+]i is required for apoptotic death. Ionomycin was used to determine the additional Ca2+ and Mg2+ mobilization potential at each dose of anti-Fas mAb (Fig. 2, C and D, open symbols). With ionomycin treatment, [Ca2+]i increased ~100 nM regardless of the dose of anti-Fas mAb used. In contrast, ionomycin caused [Mg2+]i to increase markedly only at anti-Fas mAb concentrations above 0.03 µg/ml. We used the Mg2+ response to ionomycin as a readout for cells in the high [Mg2+]i state because these data (Figs. 1C and 2, A and C) suggest that the cells that are in the high [Mg2+]i state are the same cells that further mobilize Mg2+ in response to ionomycin treatment and because the available cytometric analysis software provides a direct calculation of the percentage of cells mobilizing Mg2+ in response to ionomycin.


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Fig. 2.   The percentage of cells in the high [Mg2+]i state, which further mobilize Mg2+ upon ionomycin treatment, correlates with the percentage of cells that die by apoptosis. M12.C3 cells were treated with various concentrations of anti-Fas mAb for 16 h at 37 °C. Shown is the percentage of cells in the high [Mg2+]i state (A) or the high [Ca2+]i state (B) calculated from base-line values (before addition of ionomycin) by looking at the distribution of [Mg2+]i or [Ca2+]i at a single time point and setting gates to determine the percentage of high [Mg2+]i or high [Ca2+]i cells. The base-line levels of the mean [Mg2+]i (black-square) and [Ca2+]i () in cells incubated with increasing doses of anti-Fas mAb are indicated (C and D). After the base-line levels of [Mg2+]i and [Ca2+]i were established, the cells were treated in the cytometer with ionomycin (1 µM), and the resultant [Mg2+]i () and [Ca2+]i (open circle ) were determined. Duplicate cultures from these experiments were evaluated for apoptotic death by measuring the percentage of cells with hypodiploid levels of DNA using saponin/PI and flow cytometry (E). The maximum percentage of cells mobilizing Mg2+ at each dose of anti-Fas mAb (black-triangle) is compared with the percentage of apoptotic cells at that dose (triangle ).

Duplicate wells from the experiments shown in Fig. 2 (A-D) were examined for apoptotic cells using PI staining and flow cytometry. In saponin-permeabilized cells, PI uptake is proportional to DNA content, and apoptotic cells show diminished ("hypodiploid") staining below the G0/G1 population of viable cells (20, 24, 25). The results of this analysis indicate that the percentage of cells mobilizing Mg2+ correlates with the apoptotic (hypodiploid) cells (Fig. 2E).

Examination of Fas-mediated Apoptosis in Anti-CD40 Antibody-treated B Cells-- Incubation of murine splenic B cells with CD40 ligand leads to enhanced Fas expression and increased sensitivity to Fas-mediated apoptosis, as seen in other systems (13, 26). In a similar manner, we treated splenic B cells (which had been pretreated with anti-CD40 mAb for 72 h) with anti-Fas mAb for 2 h and then determined Mg2+ mobilization, PS externalization, and DNA fragmentation. The dose dependence of Mg2+ mobilization in anti-Fas mAb-treated B cells (Fig. 3A) was similar to that of the B cell lymphoma M12.C3 (cf. Fig. 2C). Thus, the base-line [Mg2+]i of the primary B cells also reached a plateau value of 700 µM with 0.1 µg/ml anti-Fas mAb (Fig. 3A, closed squares), and ionomycin (1 µM) treatment during the flow cytometric analysis increased [Mg2+]i 2-fold at anti-Fas mAb concentrations above 0.03 µg/ml (open squares). As with M12.C3 B cells, the percentage of cells mobilizing Mg2+ in response to ionomycin correlates with the high [Mg2+] population (data not shown).


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Fig. 3.   Examination of Fas-mediated apoptosis in CD40-stimulated B cells. Splenic B cells were obtained from AKR/J mice and incubated with anti-CD40 mAb (1C10) for 3 days. The CD40-stimulated B cells were treated with various concentrations of anti-Fas mAb as triplicate cultures for 2 h at 37 °C. The anti-Fas mAb-treated cells were loaded with mag-indo-1 and other reagents separately. A, base-line levels of the mean [Mg2+]i (black-square) in cells treated with increasing doses of anti-Fas mAb. After the base-line levels of [Mg2+]i were determined, the cells were treated in the cytometer with 1 µM ionomycin, and [Mg2+]i levels were re-determined (). B, triplicate cultures from the same experiment evaluated for apoptotic death. The percentage of cells mobilizing Mg2+ at each dose of anti-Fas mAb (black-square) was compared with the percentage of apoptotic cells as determined by DNA fragmentation (black-triangle) and PS externalization (open circle ). Curves were normalized by setting the maximum value for each parameter to 100%.

We also examined DNA fragmentation and PS externalization in the same experiment. PS is largely excluded from the external leaflet of the plasma membrane in viable cells, but becomes expressed on the external leaflet during apoptosis (5). Therefore, we used staining with annexin V, a naturally occurring PS-binding protein, to detect cells in early stages of apoptosis. Cells from parallel cultures used for Mg2+ mobilization were doubly stained with fluorescein isothiocyanate-labeled annexin V and PI, without permeabilizing the cells (5). This technique allows the definition of three subpopulations: viable cells are PS-PI-, early apoptotic cells are PS+PI-, and late apoptotic cells (and necrotic cells) are PS+PI+. In our experiments, we considered only the early apoptotic (PS+PI-) cells. The results (Fig. 3B) show that each of the responses reaches a plateau above 0.03 µg/ml anti-Fas mAb and also indicate, for each dose of mAb, that the relative percentage of cells that mobilize Mg2+ is same as the percentage of cells with increased PS externalization and DNA fragmentation. These results further demonstrate that Mg2+ mobilization is a property of primary B cells undergoing apoptosis and not just an artifact associated with the use of the B cell lymphoma M12.C3.

Kinetics of Anti-Fas mAb-induced Mg2+ Mobilization, DNA Fragmentation, and PS Externalization in B Cells-- We used kinetic analysis to determine whether Mg2+ mobilization is an early or late event in apoptosis. In M12.C3 cells treated with 0.5 µg/ml anti-Fas mAb, the mean base-line [Mg2+]i increased with time, reaching a value of 1000 µM after 1 h of incubation (Fig. 4A). At each time point (other than t = 0), ionomycin caused a significant Mg2+ mobilization in the high [Mg2+]i population of cells that were committed to die by apoptosis as previously demonstrated. The kinetics of Mg2+ mobilization versus DNA fragmentation were also measured in cells treated with 0.5 µg/ml anti-Fas mAb (Fig. 4B). The percentage of cells showing Fas-potentiated, ionomycin-induced Mg2+ mobilization was significantly higher than the percentage of those fragmenting DNA at 1-3 h after mAb treatment, but by 16 h, DNA fragmentation exceeded Mg2+ mobilization. These results suggest that Mg2+ mobilization precedes DNA fragmentation in B cells.


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Fig. 4.   Kinetics of anti-Fas mAb-induced Mg2+ mobilization, DNA fragmentation, and PS externalization in B cells. M12.C3 cells were treated with 0.5 µg/ml anti-Fas mAb at 37 °C for varying times. A, the mean [Mg2+]i was determined before (black-square) and after () treatment with ionomycin. B, comparison of the percentage of cells that mobilized Mg2+ after ionomycin treatment (black-triangle) with the percentage of apoptotic cells as determined by DNA fragmentation (down-triangle). C, comparison of the percentage of cells that mobilized Mg2+ after ionomycin treatment (black-triangle) with the percentage of apoptotic cells as determined by PS externalization (open circle ).

We decided to examine loss of plasma membrane asymmetry on these cells because, whereas DNA fragmentation is considered to be a late stage apoptotic event in many systems, PS externalization occurs early in the process. Kinetic analysis of PS externalization versus Fas-potentiated, ionomycin-mediated Mg2+ mobilization suggested that Mg2+ mobilization preceded PS externalization (Fig. 4C), although the lag in PS response was not as striking as with DNA fragmentation (Fig. 4B). Because the percentage of cells in the PS+PI- population never reached the same level of those mobilizing Mg2+, we checked to see if we were underestimating apoptosis by excluding the PS+PI+ population. The PS+PI+ population did not increase significantly over the 0-h time point except at the 16-h time point (data not shown), suggesting that the apparent decrease in PS+PI- cells at that time point may be due to their conversion to PS+PI+, late apoptotic (or necrotic) cells. However, at the earlier time points (1-3 h), loss of PS+PI- cells to the PS+PI+ population is not an explanation for lower PS+PI- numbers, and thus, PS externalization clearly lags behind Mg2+ mobilization.

Intracellular Stores Are the Primary Source of [Mg2+]i in B Cells Undergoing Apoptosis-- Calcium mobilization in B cells involves both release from intracellular stores (primarily endoplasmic reticulum) and the influx of extracellular Ca2+ (see Fig. 1 and Ref. 27). Significantly less is known about the relative roles of intracellular versus extracellular sources of Mg2+ for Mg2+ mobilization during apoptosis. We therefore examined the source of [Mg2+]i in cells undergoing apoptosis. The experiments described above were repeated with M12.C3 cells in defined media lacking Mg2+, Ca2+, or both ions. The increase in the base-line [Mg2+]i caused by anti-Fas mAb was not dependent on Mg2+ in the extracellular milieu (Fig. 5A). Likewise, the percentage of cells possessing a readily mobilizable store of Mg2+ was independent of extracellular Mg2+ (Fig. 5B). In contrast, the production of a high [Mg2+]i state and the generation of a pool of readily mobilized Mg2+ were completely dependent on extracellular Ca2+ (Fig. 5). These data support the concept that Ca2+ mobilization is required for the induction of the apoptosis signaling pathway and place the Ca2+-dependent event(s) upstream of the release of Mg2+ from intracellular stores.


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Fig. 5.   Mg2+ mobilization in B cells undergoing apoptosis does not require extracellular Mg2+, but does require extracellular Ca2+. M12.C3 cells were washed and resuspended in HBSS, either complete or modified to lack Ca2+, Mg2+, or both ions. Triplicate samples in each medium were cultured alone () or were treated for 3 h with anti-Fas mAb (). A, base-line levels of the mean [Mg2+]i of cells. B, percentage of cells with a pool of Mg2+ that can be mobilized by treatment with 1 µM ionomycin in the flow cytometer. The means ± S.E. of triplicate cultures are shown.

Inhibition of Mg2+ Mobilization and Apoptosis in B Cells by CCCP-- Mitochondria are one of the major storage sites of intracellular Mg2+ (28) and play a central role in intracellular signaling by releasing Mg2+ in response to a variety of agents (22, 29). Therefore, we investigated Mg2+ release from mitochondria during Fas-induced, ionomycin-potentiated Mg2+ mobilization using CCCP. This agent uncouples oxidative phosphorylation in mitochondria (30), and short-term treatment of cells with CCCP has been reported to disrupt Delta psi m (4). We treated M12.C3 B lymphoma cells overnight with 20 µM CCCP. Although the cells remained viable, they did show disrupted Delta psi m (as measured by decreased uptake of the cationic fluorochrome DiOC6(3); data not shown). Cells treated with CCCP for 16 h and with anti-Fas mAb for the last 3 h were examined for their ability to mobilize Mg2+ and Ca2+ in response to 1 µM ionomycin. Pretreatment of the cells with CCCP did not alter their ability to mobilize Ca2+ relative to controls not treated with CCCP (Fig. 6A, shaded bars). The slight (6%) decrease in ability to mobilize Ca2+ between cells treated with anti-Fas mAb plus CCCP and those treated with anti-Fas mAb alone was not statistically significant. These results suggest that pretreatment of the cells with CCCP did not significantly alter their ability to flux Ca2+. Conversely, whereas pretreatment of the cells with CCCP did not alter the ability of the control cells to mobilize Mg2+, it did cause a marked (40%) and highly significant decrease in the ability of the anti-Fas mAb-treated cells to mobilize Mg2+.


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Fig. 6.   Inhibition of Mg2+ mobilization and apoptosis in B cells by long-term treatment with CCCP. M12.C3 cells that had been preincubated overnight with 20 µM CCCP were treated for 3 h with 1 µg/ml anti-Fas mAb. Parallel cultures were loaded separately with mag-indo-1 or indo-1 to measure Mg2+ or Ca2+ mobilization, respectively; treated with saponin/PI to determine DNA fragmentation; or labeled with fluorescein isothiocyanate-labeled annexin V/PI to determine PS externalization. A, mean [Mg2+]i () and [Ca2+]i () produced in response to treatment with 1 µM ionomycin. Preincubation of cells with CCCP prior to anti-Fas mAb treatment did not significantly reduce Ca2+ mobilization (p < 0.46), but markedly reduced Mg2+ mobilization (p < 0.0001). B, percentage of cells mobilizing Mg2+ () in response to treatment with 1 µM ionomycin, externalizing PS (), or fragmenting DNA (black-square). Preincubation of cells with CCCP prior to anti-Fas mAb treatment caused significant reductions in Mg2+ mobilization (p < 0.0405), PS externalization (p < 0.0024), and DNA fragmentation (p < 0.0103). The means ± S.E. of triplicate cultures are shown.

In a subsequent experiment, parallel cultures were assayed for Mg2+ mobilization, PS externalization, and DNA fragmentation (Fig. 6B). In the anti-Fas mAb-treated cells, pretreatment with CCCP caused a 37% inhibition of Mg2+ mobilization relative to the untreated cells, a 41% inhibition of PS externalization, and a 45% inhibition of DNA fragmentation. In each case, the decrease in response was statistically significant, and the decreases in cells showing PS externalization and DNA fragmentation were proportional to the decrease in the number of cells mobilizing Mg2+. These data identify the mitochondria as a major source of intracellular Mg2+ and, furthermore, are consistent with a role for Mg2+ as a second messenger for apoptosis in B cells. The data also clearly separate the role of Mg2+ from that of Ca2+ since long-term treatment with CCCP does not significantly inhibit Ca2+ mobilization in response to ionomycin treatment even while Mg2+ mobilization and apoptosis are decreased.

    DISCUSSION

In this study, we have used the fluorescent Mg2+ chelator mag-indo-1 to determine [Mg2+]i. Although this chelator is the indicator of choice for determining [Mg2+]i by flow cytometry, it is capable of binding Ca2+ as well. However, other studies have indicated that [Ca2+]i in the ~1 µM range causes >7% overestimation of [Mg2+]i in the 500 µM range (31). Because the mean [Ca2+]i rarely, if ever, achieved these levels in our experiments, it is unlikely that interference from [Ca2+]i plays a significant role in our measurements of [Mg2+]i.

The role of intracellular Ca2+ in apoptosis is generally accepted (7, 32), and our studies suggest a role for elevated [Ca2+]i in B cell apoptosis as well. Multiple functions in apoptosis have been ascribed to increased [Ca2+]i. These include activation of calpain, a caspase-like protease postulated to be one of the initiators of the apoptotic cascade (33, 34); inactivation of the PS-specific flippase and activation of the phospholipid scramblase (35); and activation of Ca2+-dependent endonucleases (36-38). Mitochondria serve as sinks for Ca2+ released from the endoplasmic reticulum during intracellular Ca2+ oscillations, and it recently has been postulated that sustained elevated [Ca2+]i may cause the mitochondria to become overloaded with Ca2+, leading to downstream events that facilitate apoptosis (39). It should be noted, however, that although elevations in [Ca2+]i may be necessary for B cell apoptosis, they are not sufficient, as proliferative signals such as those produced by ligation of the B cell receptor (surface immunoglobulin) also result in Ca2+ mobilization.

In contrast to the well studied role of Ca2+ in apoptosis, the role of Mg2+ has been largely ignored. There is only a single report that elevating [Mg2+]i in hepatocytes, using glycodeoxycholates (bile salts) to allow the ingress of extracellular Mg2+, leads to their death by apoptosis (8). Intracellular Mg2+ is stored primarily in the microsomes (50% of total intracellular Mg2+) and mitochondria (20% of total), with much smaller amounts in the nucleus and cytosol (28). About 98-99% of total intracellular Mg2+ (23) and 94% of cytosolic Mg2+ (21) are complexed to nucleic acids, proteins, or membranes. Therefore, complex equilibria between compartments and between bound and free forms of Mg2+ contribute to the concentration of free cytosolic Mg2+, i.e. [Mg2+]i (28). The relatively slow release of Mg2+ (as compared with Ca2+) in the response to ionomycin treatment in our studies may reflect the need for Mg2+ not only to cross the mitochondrial membrane, but also to dissociate from immobile ligands within the mitochondria.

Because our data with the mitochondrial oxidative phosphorylation inhibitor CCCP suggest that mitochondrial stores of Mg2+ are critical for obtaining apoptosis and because our kinetic analysis suggests that Mg2+ mobilization is an early event in apoptosis, we are struck by the parallel between disruption of Delta psi m and Mg2+ mobilization. On one hand, the release of Mg2+ from the mitochondria could be merely a consequence of the opening of mitochondrial pores as Delta psi m is reduced. On the other hand, there are at least two reports that, taken together, imply that Mg2+ release from the mitochondria is independent of the loss of Delta psi m. First, the release of mitochondrial cytochrome c has been reported to occur prior to and to be independent of disruption of Delta psi m (40). Second, when isolated mitochondria are incubated with the pore-forming, pro-apoptotic molecule Bax, they incorporate Bax and release cytochrome c in a manner that is independent of Delta psi m, but "highly dependent on Mg2+ ions" (9). Because of the importance of cytochrome c release in activating apoptotic protease activating factor-1 (Apaf-1) (41, 42) and thereby driving post-mitochondrial events in cells undergoing apoptosis, we suggest that achieving high levels of [Mg2+]i is central to the apoptotic process.

Although Mg2+ is an important cofactor for a diverse set of functions in viable and proliferating cells (28), the Mg2+ dependence of a number of endonucleases implicated in apoptosis (36, 43-45) suggests that elevated Mg2+ levels are involved in apoptosis as well. The role played by Mg2+ in PS externalization is less clear. One of the best candidates for the aminophospholipid translocase ("flippase") responsible for maintaining PS asymmetry in the plasma membrane of erythrocytes is a Mg2+-ATPase (46-48). One might therefore assume that elevating [Mg2+]i should decrease PS expression on the outer leaflet of the plasma membrane. However, although the Mg2+ dependence of the flippase activity has been well documented, the effect of supernormal Mg2+ concentrations is unknown. Thus, it may be possible that elevated [Mg2+]i desensitizes the flippase, thus turning it off, or that Mg2+ has no direct effect on flippase activity and that the regulation of PS externalization by Mg2+ may be indirect, e.g. through the activation of caspases or other death-signaling molecules. Future experiments will establish the mechanism of, and the role played by, mitochondrial Mg2+ in B cells undergoing Fas-initiated apoptosis.

    ACKNOWLEDGEMENT

We thank Jennifer Vanderwall for technical and editorial assistance.

    FOOTNOTES

* This work was supported in part by United States Public Health Service Grants AI33470 and AI37523.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Div. of Rheumatology, University of Vermont, College of Medicine, Burlington, VT 05405.

parallel To whom correspondence should be addressed: Div. of Basic Immunology, National Jewish Medical and Research Center, 1400 Jackson St., Denver, CO 80206. Tel.: 303-398-1319; Fax: 303-398-1396; E-mail: freedj{at}njc.org.

    ABBREVIATIONS

The abbreviations used are: mAb, monoclonal antibody; Delta psi m, mitochondrial membrane potential; PS, phosphatidylserine; [Ca2+]i, free cytosolic Ca2+ concentration; [Mg2+]i, free cytosolic Mg2+ concentration; HBSS, Hanks' balanced salt solution; PI, propidium iodide; CCCP, carbonyl cyanide m-chlorophenylhydrazone.

    REFERENCES
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