HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by He, H. Articles by McCarthy, S. A. Search for Related Content PubMed PubMed Citation Articles by He, H. Articles by McCarthy, S. A.

Down-Modulation of a Novel Bax-Associated Protein During Apoptosis in Normal Mature B Lymphocytes¹

Huiling He^2,*, Pamela A. Hershberger^3,* and Susan A. McCarthy^4,*,

Departments of ^* Surgery and Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have recently characterized a novel 16-kDa Bax-associated protein. In this study, we investigate the regulation of this protein’s expression during in vitro induction of apoptosis in mature splenic B cells. A panel of biochemically distinct apoptotic stimuli induced the dramatic down-modulation of the 16-kDa protein in B cells; this down-modulation was rapid, and did not require DNA fragmentation. Reciprocally, stimuli that induced protection from apoptosis prevented down-modulation of the 16-kDa protein. These regulatory effects were specific, since Bcl-2 and Bax protein levels were not similarly modulated. Stimuli that reduce expression of the 16-kDa protein may therefore act indirectly to increase the proapoptotic activity of Bax, perhaps by altering Bax binding to other cellular proteins.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Apoptosis is an active process resulting in cell suicide. In the immune system, apoptosis plays a pivotal role in the selection of lymphocyte populations and maintenance of functional immune responses. Apoptosis is a prominent feature during B cell differentiation and occurs at several maturation stages. Immature B cells that either fail to express functional IgM molecules or express IgM receptors with high affinity for self-Ags are deleted by apoptotic cell death (1). During an immune response, an initial clonal expansion of mature Ag-specific B cells is followed by elimination of the vast majority of those cells by apoptosis (2). An understanding of the molecular events and regulatory mechanisms controlling apoptosis, as well as resistance to apoptosis, of normal mature B cells is therefore critical for therapeutic strategies designed to manipulate the B cell response to Ags.

A family of related proteins, including the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax, has been identified as having important regulatory roles in B cell apoptosis (reviewed in Refs. 3 and 4). Bcl-2 was first discovered by virtue of its involvement in the t(14;18) chromosomal translocation found in the majority of non-Hodgkin’s B cell lymphomas (5, 6, 7). Later in vitro studies demonstrated that Bcl-2 overexpression, which mimics the consequence of the t(14:18) translocation, prevents cell death (8) and may thereby increase a cell’s tumorigenic potential. In agreement with these in vitro findings is the observation that transgenic mice with enforced overexpression of Bcl-2 in the B cell lineage develop B cell hyperplasia that may progress to malignant lymphomas (9). More recent studies implicate a homozygous deficiency in Bcl-2 expression in the elimination of mature B lymphocytes and/or B cell precursors due to enhanced apoptosis (10, 11), indicating that natural, endogenous Bcl-2 levels are sufficient to promote the survival of normal, untransformed B cells. Taken together, these studies provide evidence that regulation of Bcl-2 expression and function is important for B cell homeostasis.

Bax was originally identified as a protein that heterodimerizes with Bcl-2 in vitro and accelerates cell death when overexpressed (12). Consistent with Bax being a death-promoting molecule in lymphocytes, primary T cells isolated from Bax transgenic animals displayed accelerated cell death in response to a variety of apoptosis-inducing stimuli, as compared with nontransgenic controls (13). Moreover, the demonstration that a homozygous deficiency in Bax expression results in lymphoid hyperplasia (14) indicates that natural, endogenous Bax levels are sufficient to negatively regulate the life span of normal T and B lymphocytes. Thus, like Bcl-2, Bax clearly plays a role in regulating lymphocyte homeostasis.

More recent studies have identified additional Bcl-2 family members that belong to either an antiapoptotic functional subset (such as Bcl-XL (15)) or a proapoptotic subset (such as Bad (16)), which regulate cell death via their heterodimerization with Bcl-2 and/or Bax (reviewed in 3 . In the accompanying manuscript, we describe a novel 16-kDa Bax-associated protein (referred to here as "P16," based on the approximate size of the protein and not meant to imply any relationship to the cell cycle-associated p16 protein) in which expression is down-modulated during thymocyte apoptosis (17). Based on these properties, we propose that P16 may represent another member of the Bcl-2 family of apoptotic regulators.

Since Bcl-2, Bax, and other family member proteins homo- and heterodimerize in vivo, it has been proposed that the ratio of proapoptotic to antiapoptotic proteins (the ratio of Bax to Bcl-2, for example) in a cell is critical to the fate of the cell upon exposure to an apoptotic stimulus. This hypothesis has been supported by the kinds of transgenic and transfection studies outlined above, in which deliberate manipulation of the Bax to Bcl-2 ratio alters cellular responsiveness to apoptotic stimuli. In the present study, we have approached this hypothesis from the reciprocal perspective and have investigated whether stimuli that induce apoptosis, or resistance to apoptosis, in normal mature B cells do so by regulating the expression of pro- and/or antiapoptotic proteins within the cells. We demonstrate that whereas a number of apoptosis-regulating stimuli have no corresponding effect on expression of Bcl-2 and Bax, their effects on P16 expression are dramatic: proapoptotic stimuli rapidly down-modulate P16 expression, and antiapoptotic stimuli preserve P16 expression. These results, together with those presented in the accompanying paper (17), are consistent with the hypothesis that P16 represents a new antiapoptotic protein expressed in lymphocytes that may act by heterodimerizing with Bax and neutralizing its proapoptotic activity.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

Female C57BL/6J (B6)⁵ mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were used between 7 and 13 wk of age.

Abs and chemicals

The anti-CD4 mAb RL172 was used as a tissue culture supernatant, and the anti-CD8 mAb 83-12-5 was from ascites fluid. Polyclonal anti-mouse Bax Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-E2 Ab (designated N20 by Santa Cruz) is an anti-Bax Ab raised against a 19-amino acid peptide corresponding to residues 11–30 of the Bax protein, encoded by exon 2 of the Bax gene. Anti-E3 Ab (designated P19 by Santa Cruz) is an anti-Bax Ab raised against a 19-amino acid peptide corresponding to residues 43–61 of the Bax protein, encoded by exon 3 of the Bax gene. The hamster monoclonal anti-Bcl-2 Ab 3F11 was kindly provided by Dr. Stanley Korsmeyer (Washington University, St. Louis, MO) or was purchased from PharMingen (San Diego, CA). Goat anti-rabbit IgG conjugated with horseradish peroxidase and anti-hamster IgG conjugated with biotin were purchased from Sigma Chemical (St. Louis, MO) and PharMingen, respectively. Rabbit complement was purchased from Cedarlane Laboratories (Hornby, Ontario, Canada). A23187, PMA, dexamethasone (Dex),⁵ actinomycin D (Act D), and cycloheximide (CHX) were all purchased from Sigma Chemical and were stored as concentrated stock solutions at -30°C. Cyclosporin A (CsA; lot OL27-400; a gift from Dr. W. Houlihan, Sandoz Research Institute, East Hanover, NJ) was diluted to 1 mg/ml in 5:1 ethanol:Tween 80 and stored at -30°C. IL-4 was purchased from R & D Systems (Minneapolis, MN) and was stored as a concentrated stock solution at -80°C. Genistein was purchased from Calbiochem-Novabiochem (La Jolla, CA). The caspase inhibitor zVAD.CH2F and the control reagent zFA-CH2F were purchased from Enzyme Systems Products (Livermore, CA), stored at 4°C, and diluted in tissue culture medium for use.

Splenocyte isolation and B cell purification

Splenocytes were prepared by mechanical disruption of freshly isolated B6 spleens. Splenocytes were treated briefly with ACK (18) to lyse contaminating erythrocytes. Splenocyte suspensions were then filtered through nylon mesh to remove cell aggregates and washed. Mature B cells were purified from washed splenocyte suspensions by complement depletion of CD4⁺ and CD8⁺ T cells. Briefly, 1 x 10⁷ splenocytes/ml were incubated with saturating concentrations of anti-CD4 and anti-CD8 mAbs at 4°C for 30 min. The anti-CD4- and anti-CD8-coated cells were collected by centrifugation, incubated with rabbit or guinea pig complement at 37°C for 40 min, and then washed three times. Washed cells were resuspended in fresh RPMI medium supplemented with 5% FCS, 2-ME, glutamine, sodium pyruvate, nonessential amino acids, penicillin and streptomycin and cultured at a density of 2.5 x 10⁶ cells/ml at 37°C in an atmosphere containing 7.5% CO₂. To evaluate B cell purity, flow cytometric analysis was routinely done following each T cell depletion. These analyses revealed that B cells (B220⁺CD3^-) were consistently obtained at >90% purity; in our hands, splenic B200⁺ cells from normal adult mice typically contain approximately 5% B220⁺IgM^-IgD^- progenitor cells, 10% B220⁺IgM⁺ IgD^- immature B cells, and 85% B220⁺IgM⁺IgD⁺ mature B cells.

DNA fragmentation assay

DNA fragmentation assays were performed as previously described (18). Briefly, 5.0 x 10⁶ B cells were incubated in 2 ml of tissue culture medium in the presence of the indicated stimuli at 37°C for varying lengths of time. Stimuli were used at the following final concentrations: A23187 (100 ng/ml), CsA (12.5 ng/ml), PMA (10 ng/ml), Dex (1 µM), IL-4 (1 ng/ml), Act D (1.0 µg/ml), CHX (10 µg/ml), genistein (5 µg/ml), zVAD.CH2F (20 µM), and zFA.CH2F (20 µM). After culture, the cells (1 x 10⁷ per experimental group) were collected, washed three times in PBS, and lysed for 30 min at 4°C in a buffer containing 5 mM Tris, pH 8.0, 1 mM EDTA, and 0.5% Triton X-100. Fragmented and intact DNA were separated by centrifugation at 13,200 rpm at 4°C. Fragmented DNA (supernatant) and intact DNA (pellet) fractions were adjusted to the same final volume in the lysis buffer. Each sample was then sonicated, and the samples were plated in triplicate 100-µl serial dilutions in Dynatech MicroFluor 96-well plates (Dynatech Labs, Alexandria, VA). One hundred microliters of a solution of 0.6 mg/ml of the fluorescent DNA dye, DAPI (4,6-diamidino-2-phenylindole) was then added to each well. The relative amount of DNA in each sample was derived from the fluorescence emission at 465 nm, as measured on a Dynatech MicroFluor plate reader. The percentage of DNA fragmentation was calculated as: % fragmentation = 100 x (DNA in supernatant/(DNA in supernatant + DNA in pellet)).

Immunoblotting

After culture with the indicated stimuli, the B cells were harvested, washed twice with cold PBS, and then lysed at 4°C for 40 min in a lysis buffer containing 1% Triton-X100, 0.1% SDS, 50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1 mM PMSF, and 1 µg/ml leupeptin. The lysates were spun in a microfuge at 13,200 rpm for 10 min, the supernatants collected, and the protein concentrations determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA). Thirty micrograms of each protein lysate was electrophoresed on 12 or 12.5% polyacrylamide gels under reducing conditions (5% 2-ME added) and electrophoretically transferred to either nitrocellulose membranes (Amersham Life Science, Arlington Heights, IL) or polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were incubated in 5% nonfat dry milk in T-TBS (18 mM Tris-Cl pH 7.6, 122 mM NaCl, 0.1% Tween 20) at room temperature for at least 2 h to minimize nonspecific binding of Ab. The membranes were then incubated with the indicated primary Abs at 4°C overnight, then incubated with secondary Ab at room temperature for 1 h. Immune complexes were detected with the Renaissance chemiluminescence reagent (DuPont-NEN, Boston, MA) by treating the membranes according to the manufacturer’s protocol, followed by exposure to x-ray film (Sigma).

Densitometry

The relative protein expression levels of P16, Bax, and Bcl-2 were quantitated by densitometry (Molecular Dynamics, Sunnyvale, CA) of immunoblots.

Statistics

Statistical analyses used the Student’s t test; p 0.05 is considered a statistically significant difference between values.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of B cell apoptosis and differential protection by IL-4

Mature splenic murine B cells can be induced, in vitro, to undergo apoptosis and can be protected from induction of apoptosis by a number of biochemically distinct stimuli. We have previously used a series of such proapoptotic and antiapoptotic (protective) stimuli to characterize the regulation of apoptosis in normal mature B cells (manuscript in preparation). In the current study, we cultured enriched splenic B cells for 21 h with the calcium ionophore A23187 plus CsA (A23 + CsA) or with the steroid Dex; or we exposed the B cells to gamma radiation (Rad) before culture (Fig. 1). Each of these stimuli induced high and comparable levels of apoptosis as measured by a quantitative DNA fragmentation assay. In addition, we tested each of the apoptotic stimuli in the presence of the T cell-derived cytokine IL-4, which we and others have shown can inhibit B cell apoptosis in response to some stimuli (Fig. 1A). IL-4 completely inhibited B cell apoptosis in response to A23 + CsA, but only partially inhibited B cell apoptosis in response to Dex, and did not inhibit B cell apoptosis in response to Rad (Fig. 1A). Thus, these eight test conditions represent a panel of nonapoptotic, apoptotic, and antiapoptotic stimuli for analysis of Bcl-2 family protein expression.

View larger version (53K): [in this window] [in a new window]   FIGURE 1. Bcl-2 and Bax levels are not modulated during mature B cell apoptosis. B6 enriched splenic B cells were cultured for 21 h in medium (Med), with A23 + CsA, or with Dex, or they were exposed to 500R Rad before culture in Med; each stimulation group also was cultured with or without IL-4, as indicated. In addition, one aliquot of cells was maintained in Med at 4°C for the 21-h culture period as a control group. After culture, the percentage of DNA fragmentation was determined for each cell group (A). A representative experiment is shown in A. After culture, the lysate of a second aliquot from each culture group was separated by SDS-PAGE under reducing conditions and immunoblotted with anti-Bcl-2 Ab (B) and anti-E2 Bax Ab (C). The relative Bcl-2 and Bax protein expression levels were quantitated by densitometry, with the Med control group’s protein level for each protein set at 100. The expression levels in the treatment groups were calculated relative to the Med group for each experiment. B and C are from three and two independent experiments, respectively, each with all nine treatment groups. No statistically significant changes in Bcl-2 or Bax expression, relative to the Med control, were observed among the eight treatment groups.

We first analyzed whether the panel of stimuli regulate B cell apoptosis by regulating the expression of the best-characterized Bcl-2 family members, Bcl-2 itself (which has antiapoptotic activity) and Bax (which has proapoptotic activity). After a 21-h stimulation period, one aliquot of each cell group was used for quantitation of DNA fragmentation, as in Figure 1A, and a second aliquot was used for quantitation of protein expression by Western blot analysis. Analyses were performed on equal amounts of total protein from each treatment group, so that any change in a protein’s expression level reflects a specific modulation of that protein and not an overall change in protein level in the cells. Figure 1B illustrates that the Bcl-2 protein level was not modulated by any of the stimuli, despite their pronounced effects on levels of apoptosis. We also analyzed cultures stimulated for 4 and 12 h and again detected no modulation of Bcl-2 protein levels (data not shown). To exclude the possibility that Bcl-2 levels are up-modulated on some cells and down-modulated on other cells within a culture, resulting in no net change in total Bcl-2 level in the culture, we used FACS analysis of Bcl-2 on permeablized cells. We found that cells from each of the eight stimulation conditions were quite homogeneous for Bcl-2 expression, with no evidence of modulation by the pro- and antiapoptotic stimuli (data not shown). Taken together, these results demonstrate that Bcl-2 protein level is not down-modulated during the course of apoptosis in mature splenic B cells and that the protective effect of IL-4 on these cells is not mediated by up-modulation of Bcl-2 protein levels.

Figure 1C illustrates that regulation of Bax expression also does not correlate with regulation of apoptosis in mature splenic B cells. A23 + CsA and Rad, which induced extensive apoptosis in these cells, did so without any up-modulation in Bax expression. Dex, which also induced extensive apoptosis, did so despite a modest decrease in Bax protein expression. Finally, IL-4, which differentially protected the B cells from apoptosis, did so without down-modulating Bax expression. We have observed the same pattern of Bax expression using a different anti-Bax Ab in the Western analysis (data not shown). Thus, we found no evidence that apoptotic stimuli act by increasing Bax expression or that antiapoptotic stimuli act by decreasing Bax expression in normal mature B cells.

A Bax-associated protein, P16, is down-modulated during mature B cell apoptosis

In the accompanying manuscript, we describe a 16-kDa protein (P16) that can be detected with an anti-Bax Ab in normal and Bax knock-out murine lymphocytes and that associates with Bax intracellularly in normal lymphocytes (17). In thymocytes, P16 is down-modulated in response to two apoptotic stimuli (see accompanying paper (17)), suggesting that it might contribute to the regulation of apoptosis. We therefore analyzed P16 protein levels in mature B cells, using the panel of apoptotic and antiapoptotic stimuli. In contrast to Bcl-2 and Bax, P16 was highly regulated by these stimuli. P16 protein levels were dramatically down-modulated by all three of the apoptotic stimuli (Fig. 2). Conversely, P16 protein levels were preserved by IL-4, but only in combinations in which IL-4 substantially inhibited apoptosis. For example, in combination with A23 + CsA, IL-4 completely inhibited apoptosis and preserved P16 protein levels, whereas in combination with Rad, IL-4 failed to inhibit apoptosis and failed to preserve P16 protein levels. Thus, within this panel of eight biochemically distinct stimulation conditions, P16 protein levels were inversely correlated with the levels of B cell apoptosis, supporting the hypothesis that P16 functions as an antiapoptotic protein that must be eliminated in order for apoptosis to proceed.

View larger version (50K): [in this window] [in a new window]   FIGURE 2. P16 is down-modulated during mature B cell apoptosis. B6 enriched splenic B cells were cultured for 21 h in medium (Med), with A23 + CsA, or with Dex, or they were exposed to 500R Rad before culture in Med; each stimulation group also was cultured with or without IL-4, as indicated. In addition, one aliquot of cells was maintained in medium at 4°C for the 21 h culture period as a control group. After culture, one aliquot for each treatment group was collected and used to determine percent DNA fragmentation. A second aliquot was separated by SDS-PAGE under reducing conditions and immunoblotted with anti-E3 Bax Ab. The relative P16 protein expression levels were quantitated by densitometry, with the Med control group’s protein level set at 100. The expression level in the treatment groups was calculated relative to the Med control group for each experiment (mean ± SD). A representative experiment is shown for the Western blot and its associated DNA fragmentation; the densitometry data are from three independent experiments. Statistically significant reductions in P16 expression were observed for the A23 + CsA, Dex, and Rad treatment groups, relative to the Med control group. Sparing of P16 expression by IL-4 was statistically significant only for the A23 + CsA + IL-4 treatment group, relative to the Med control group.

View larger version (54K): [in this window] [in a new window]   FIGURE 3. P16 is down-modulated during mature B cell apoptosis induced by a variety of stimuli. B6 enriched splenic B cells were cultured for 21 h in medium (Med) or with the indicated stimuli. After culture, one aliquot for each treatment group was collected and used to determine percentage of DNA fragmentation. A second aliquot was separated by SDS-PAGE under reducing conditions and immunoblotted with anti-E3 Bax Ab. A representative experiment is shown for each stimulus.

P16 protein down-modulation was directly correlated with induction of DNA fragmentation in response to all of the stimuli tested. To investigate whether P16 reduction is a cause or a consequence of DNA fragmentation and subsequent cell death, we analyzed the kinetics of P16 reduction and DNA fragmentation (Fig. 4). B cells were treated with A23 + CsA, Dex, or Rad for varying time periods before quantitation of P16 down-modulation and DNA fragmentation. For each of the three apoptotic stimuli, P16 down-modulation preceded DNA fragmentation, although the interval between these two processes was not dramatic and varied somewhat among the stimuli. This result suggested that P16 down-modulation might be up-stream of DNA fragmentation in the apoptotic cascade.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. P16 down-modulation is an early event during mature B cell apoptosis. B6 enriched splenic B cells were cultured for 3 to 21 h in medium (Med) with A23 + CsA, or with Dex, or they were exposed to 500R Rad before culture in Med. In addition, one aliquot of cells was maintained in medium at 4°C for the 21-h culture period as a control group. At each of the indicated time points, one aliquot for each treatment group was collected and used to determine percent DNA fragmentation. A second aliquot was separated by SDS-PAGE under reducing conditions and immunoblotted with anti-E3 Bax Ab. The relative P16 protein expression levels were quantitated by densitometry, with the 4°C control culture’s protein level set at "0 P16 reduction." The P16 reduction in the treatment groups was calculated relative to the 4°C control culture. A representative experiment is shown.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. P16 down-modulation does not require DNA fragmentation. B6 enriched splenic B cells were cultured for 21 h in medium (Med) or with Dex, or they were exposed to 500R Rad before culture in Med; each stimulation group also was cultured with or without the caspase inhibitor, zVAD.CH2F, as indicated. After culture, one aliquot for each treatment group was collected and used to determine percentage of DNA fragmentation. A second aliquot was separated by SDS-PAGE under reducing conditions and immunoblotted with anti-E3 Bax Ab. The relative P16 protein expression levels were quantitated by densitometry, with the medium control culture’s protein level set at "0 P16 reduction" (indicated by an asterisk (*) in the top panel). The P16 reduction in the treatment groups was calculated relative to the medium control culture. A representative experiment is shown.

P16 protein levels are dramatically reduced in apoptotic B cells, which suggests that P16 protein may be required for B cell survival. In previous studies (manuscript in preparation), we had found that IL-4 added to B cells at a relatively late time point (12 h into a 20-h culture) can substantially inhibit DNA fragmentation induced by A23 + CsA. We therefore tested whether late addition of IL-4 spares B cells from apoptosis by restoring P16 levels to those seen in nonapoptotic cells (Fig. 6). After initiation of the culture in the presence of A23 + CsA, IL-4 was added at 12 h, and the B cells were cultured for an additional 8 h. As expected, IL-4 added at 12 h inhibited further DNA fragmentation, so that the DNA fragmentation measured at 20 h in cultures that had received IL-4 at 12 h was very similar to the DNA fragmentation measured at 12 h. However, IL-4 added at 12 h did not reverse or inhibit P16 down-modulation, with the consequence that at 20 h the B cells had dramatically down-modulated their P16 but had nevertheless been substantially protected from DNA fragmentation by IL-4. These results suggest that IL-4 can affect the apoptotic cascade at at least two points: before P16 down-modulation (when IL-4 is added at culture initiation; Fig. 2); and after P16 down-modulation (when IL-4 addition is delayed; Fig. 6). These results also demonstrate that normal P16 protein levels are not always required for B cell protection from apoptosis, if an antiapoptotic stimulus such as IL-4 is provided. Although we have identified one situation in which B cells are protected from apoptosis despite P16 down-modulation (Fig. 6), we have not identified any situation in which B cells are induced to undergo apoptosis without P16 down-modulation. Thus, P16 down-modulation may be necessary but not sufficient for induction of apoptosis in mature splenic B cells.

View larger version (18K): [in this window] [in a new window]   FIGURE 6. Normal P16 expression levels are not required for B cell protection from apoptosis. B6 enriched splenic B cells were cultured for 12 or 20 h in medium (Med) or with A23 + CsA (A+C), as indicated; IL-4 was added to some cultures after 12 h, as indicated. In addition, one aliquot of cells was maintained in medium at 4°C for the 21-h culture period as a control group. At the indicated time points, one aliquot for each treatment group was harvested and used to determine percentage of DNA fragmentation. A second aliquot was separated by SDS-PAGE under reducing conditions and immunoblotted with anti-E3 Bax Ab. The relative P16 protein expression levels were quantitated by densitometry, with the 4°C control culture’s protein level set at "0 P16 reduction". The P16 reduction in the treatment groups was calculated relative to the 4°C control culture. A representative experiment is shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Regulation of apoptosis by Bcl-2 family members

A large body of evidence now exists supporting the idea that cell death is regulated by homodimerization and/or heterodimerization among proapoptotic and antiapoptotic Bcl-2 family members (reviewed in 3 . In the simplest interpretation, the available data indicate that enforced expression of Bax, which leads to a predominance of Bax:Bax homodimers, promotes cell death (12). Conversely, overexpression of Bcl-2 (or Bcl-XL), which would be expected to favor the generation of Bcl-2:Bcl-2 homodimers or Bax:Bcl-2 heterodimers and to reduce the levels of Bax:Bax homodimers, does indeed protect from apoptosis (21). Furthermore, mutation of such overexpressed Bcl-2 to a form that cannot heterodimerize with Bax fails to protect from apoptosis (21). Based on such studies, it can be hypothesized that apoptotic stimuli normally regulate cell death either by reducing the level of endogenous antiapoptotic family members (Bcl-2 or Bcl-XL) or by increasing the level of proapoptotic family members (Bax). Some studies have, in fact, demonstrated a correlation between increased lymphocyte apoptosis and down-modulation of endogenous Bcl-2 expression during HIV infection (22).

Based on findings such as the preceding, it was somewhat surprising to find that a panel of distinct biochemical stimuli could induce apoptosis in normal B lymphocytes without modulating Bcl-2 or Bax levels. Instead, our data indicate that the apoptotic pathway in these cells is triggered by reducing the levels of the Bax-binding partner, P16. Extrapolating from the above model of dimerization among family members, a reduction in P16 levels would reduce the number of Bax:P16 complexes. This may favor the formation of proapoptotic Bax:Bax homodimers, resulting in the induction of cell death. However, the actual influence of P16 reduction on intracellular Bax associations remains to be determined. Others have also reported that apoptosis may be induced independent of modulation of endogenous Bcl-2 expression. For example, others have shown that Bcl-2 levels are not dramatically altered in hemopoietic cells induced to undergo apoptosis upon IL-3 withdrawal (23). Instead, it appears that IL-3 withdrawal results in dephosphorylation of the distantly related Bcl-2 family member, BAD (24). BAD dephosphorylation results in the preferential association of BAD with Bcl-XL (24), which may in turn cause the release of Bax from Bax:Bcl-XL heterodimers leading to the formation of Bax:Bax homodimers and cell death.

What is P16 and where does it function in the apoptotic cascade in B cells?

Based on the facts that P16 is recognized by an Ab raised against an epitope encoded by exon 3 of Bax and associates with Bax in normal lymphocytes (see accompanying paper (17)), we propose that P16 is likely to minimally encode a BH3, Bcl-2 homology domain. Ab mapping studies suggest that P16 may share no other antigenic epitopes with Bax outside of that encoded by exon 3 (unpublished observation). Collectively, these findings preliminarily place P16 into an expanding class of distantly related Bcl-2 family members that encode only a BH3 motif (25, 26). The cloning of P16, which is currently in progress, will test this hypothesis.

We observed that P16 down-modulation in B cells is not blocked by the caspase inhibitor, zVAD.CH2F (Fig. 5), although this inhibitor does prevent DNA fragmentation in these cells. One interpretation of this result is that during B cell apoptosis, P16 down-modulation occurs before caspase activation and therefore is not susceptible to inhibition by zVAD.CH2F. This interpretation would suggest that P16 normally exerts its antiapoptotic function in B cells before the activation of caspases. A protease-proximal function for P16 in B cell apoptosis lends further support to the hypothesis that P16 is at least a distant member of the Bcl-2 family, since biochemical studies indicate that Bcl-2 functions upstream of caspase activation (27, 28). At the current time, however, we cannot eliminate the alternative hypothesis for the lack of inhibition of P16 down-modulation by zVAD.CH2F in which P16 down-modulation occurs by a pathway parallel to that resulting in caspase activation.

IL-4 protects B cells from apoptosis via P16-dependent and P16-independent mechanisms

IL-4 is a known B cell growth factor and has been shown to protect immature and mature B cells from apoptosis induced by multiple stimuli (1, 29, 30). However, little is known about the mechanism by which IL-4 protects B cells from apoptosis. To investigate the molecular basis for IL-4 protection, we have examined the effects of IL-4 addition on the endogenous expression of Bcl-2 family members. IL-4 significantly protected B cells from apoptosis induced by A23 + CsA (Fig. 1). Although A23 + CsA alone induced a dramatic down-modulation of P16, P16 levels were preserved in cells treated with A23 + CsA + IL-4 (Fig. 2). This result suggests that one mechanism by which IL-4 protects B cells from apoptosis is by maintaining their expression of P16.

IL-4 clearly also functions to protect B cells by a second P16-independent mechanism, since IL-4 can afford protection from apoptosis in B cells in which P16 down-modulation occurs (Fig. 6). Interestingly, a recent report also defines two separate mechanisms by which IL-4 protects B cells from apoptosis; one that is dependent on the insulin receptor substrate/phosphatidylinositol-3'-kinase (IRS/PI3K) pathway and one that is independent of this pathway (31). However, the relationship between IL-4 protection pathways that are P16-dependent or P16-independent and IL-4 protection pathways that are IRS/PI3K-dependent and IRS/PI3K-independent remains to be determined.

Conclusion

We have characterized a 16-kDa Bax-associated protein whose expression is rapidly down-modulated in normal B cells (this study) and thymocytes (accompanying paper (17)) induced to undergo apoptosis. We propose that this protein provides an antiapoptotic function that normally must be abrogated for apoptosis to proceed in these cells. The biochemical diversity of the stimuli mediating P16 down-modulation leads us to further suggest that P16 is positioned at a point in the lymphocyte apoptotic cascade at which multiple signaling pathways converge. This property makes P16 an attractive target for therapeutic intervention in diseases resulting from aberrant lymphocyte apoptosis.

	Acknowledgments

 
We thank Drs. M. Tector, R. Salter, T. Wright, D. Johnson, and C. Brock for reagents, discussions, and critiques of the manuscript.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant R29AI32554 (S.McC.); a grant from the Arthritis Foundation, Western Pennsylvania Chapter (S.McC.); American Cancer Society Grant IRG-58-35 (H.H.); and National Institutes of Health/National Research Service Award Grant F32AI09634 (P.A.H.).

² Current address: Dr. Huiling He, Department of Medicine, School of Medicine (BRB), Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106.

³ Current address: Dr. Pamela A. Hershberger, Department of Pharmacology, W1002 Biomedical Science Tower, Terrace and Lothrop Streets, University of Pittsburgh, Pittsburgh, PA 15213.

⁴ Address correspondence and reprint requests to Dr. Susan A. McCarthy, Department of Surgery, W1554 Biomedical Science Tower, Terrace and Lothrop Streets, University of Pittsburgh, Pittsburgh, PA 15213. E-mail address:

⁵ Abbreviations used in this paper: Dex, dexamethasone; Act D, actinomycin D; B6, C57BL/6J; CHX, cycloheximide; CsA, cyclosporin A; A23 + CsA, calcium ionophore A23187 + cyclosporin A; IRS/PI3K, insulin receptor substrate/phosphatidylinositol-3'-kinase; Rad, gamma radiation.

Received for publication January 21, 1998. Accepted for publication March 31, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Norvell, A., L. Mandik, J. G. Monroe. 1995. Engagement of the antigen-receptor on immature B lymphocytes results in death by apoptosis. J. Immunol. 154:4404.[Abstract]
2. Shokat, K. M., C. C. Goodnow. 1995. Antigen-induced B-cell death and elimination during germinal-centre immune responses. Nature 375:334.[Medline]
3. Yang, E., S. J. Korsmeyer. 1996. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 88:386.[Free Full Text]
4. Reed, J. C.. 1997. Double identity for proteins of the Bcl-2 family. Nature 387:773.[Medline]
5. Bakhshi, A., J. P. Jensen, P. Goldman, J. J. Wright, O. W. McBride, A. L. Epstein, S. J. Korsmeyer. 1985. Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 near a transcriptional unit on 18. Cell 41:889.
6. Cleary, M. L., J. Sklar. 1985. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint cluster near a transcriptionally active locus on chromosome 18. Proc. Natl. Acad. Sci. USA 82:7439.[Abstract/Free Full Text]
7. Tsujimoto, Y., J. Cossman, E. Jaffe, C. M. Croce. 1985. Involvement of the bcl-2 gene in human follicular lymphoma. Science 228:1440.[Abstract/Free Full Text]
8. Hockenberry, D., G. Nunez, C. Milliman, R. D. Schreiber, S. J. Korsmeyer. 1990. Bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death. Nature 348:334.[Medline]
9. McDonnell, T. J., S. J. Korsmeyer. 1991. Progression from lymphoid hyperplasia to high-grade malignant lymphoma in mice transgenic for the t(14;18). Nature 349:254.[Medline]
10. Nakayama, K., K. Nakayama, I. Negishi, K. Kuida, Y. Shinkai, M. C. Louie, L. E. Fields, P. J. Lucas, V. Stewart, F. W. Alt, D. Y. Loh. 1993. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science 261:1584.[Abstract/Free Full Text]
11. Veis, D. J., C. M. Sorenson, J. R. Shutter, S. J. Korsmeyer. 1993. Bcl-2-deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys, and hypopigmented hair. Cell 75:229.[Medline]
12. Oltvai, Z. N., C. L. Milliman, S. J. Korsmeyer. 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 74:609.[Medline]
13. Brady, H. J. M., G. S. Salomons, R. C. Bobeldijk, A. J. M. Berns. 1996. T cells from bax transgenic mice show accelerated apoptosis in response to stimuli but do not show restored DNA damage-induced cell death in the absence of p53. EMBO J. 15:1221.[Medline]
14. Knudson, C. M., K. S. K. Tung, W. G. Tourtellotte, G. A. J. Brown, S. J. Korsmeyer. 1995. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 270:96.[Abstract/Free Full Text]
15. Boise, L. H., M. Gonzalez-Garcia, C. E. Postema, L. Ding, T. Lindsten, L. A. Turka, X. Mao, G. Nunez, C. B. Thompson. 1993. Bcl-x, a bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74:597.[Medline]
16. Yang, E., J. Zha, J. Jockel, L. H. Boise, C. B. Thompson, S. J. Korsmeyer. 1995. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 80:285.[Medline]
17. He, H., P. A. Hershberger, S. A. McCarthy. 1998. Characterization of a novel Bax-associated protein expressed in hemopoietic tissues and regulated during thymocyte apoptosis. J. Immunol. 161:1176.[Abstract/Free Full Text]
18. Coligan, J. E., A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober (eds). 1991. Current Protocols in Immunology. Greene Publishing Associates and Wiley Interscience, New York, pp. 3.1.3–3.1.5 and 8.1.1–8.1.6.
19. McCarthy, S. A., R. N. Cacchione, M. S. Mainwaring, J. S. Cairns. 1992. The effects of immunosuppressive drugs on the regulation of activation-induced apoptotic cell death in thymocytes. Transplantation 54:543.[Medline]
20. Henkart, P.. 1996. ICE family proteases: mediators of all apoptotic cell death?. Immunity 4:195.[Medline]
21. Yin, X-M., Z. N. Oltvai, S. J. Korsmeyer. 1994. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 369:321.[Medline]
22. Strack, P. R., M. W. Frey, C. J. Rizzo, B. Cordova, H. J. George, R. Meade, S. P. Ho, J. Corman, R. Tritch, R. D. Korant. 1996. Apoptosis mediated by HIV protease is preceded by cleavage of Bcl-2. Proc. Natl. Acad. Sci. USA 93:9571.[Abstract/Free Full Text]
23. Canman, C. E., T. M. Gilmer, S. B. Coutts, M. B. Kastan. 1995. Growth factor modulation of p53-mediated growth arrest vs apoptosis. Genes Dev. 9:600.[Abstract/Free Full Text]
24. Zha, J., H. Harada, E. Yang, J. Jockel, S. J. Korsmeyer. 1996. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3, not Bcl-XL. Cell 87:619.[Medline]
25. Wang, K., X.-M. Yin, D. T. Chao, C. L. Milliman, S. J. Korsmeyer. 1996. Bid: a novel BH3 domain-only death agonist. Genes Dev. 10:2859.[Abstract/Free Full Text]
26. Inohara, N., L. Ding, S. Chen, G. Nunez. 1997. Harakiri, a novel regulator of cell death, encodes a protein that activates apoptosis and interacts selectively with survival-promoting proteins Bcl-2 and Bcl-XL. EMBO J. 16:1686.[Medline]
27. Boulakia, C. A., G. Chen, F. W. H. Ng, J. G. Teodoro, P. E. Branton, D. W. Nicholson, G. G. Poirier, G. C. Shore. 1996. Bcl-2 and adenovirus E1B 19 kDa protein prevent E1A-induced processing of CPP32 and cleavage of poly(ADP-ribose)polymerase. Oncogene 12:529.[Medline]
28. Chinnaiyan, A. M., K. Orth, K. O’Rourke, H. Duan, G. G. Poirier, V. M. Dixit. 1996. Molecular ordering of the cell death pathway. Bcl-2 and Bcl-XL function upstream of the ced-3-like apoptotic proteases. J. Biol. Chem. 271:4573.[Abstract/Free Full Text]
29. Illera, V. A., C. E. Perandones, L. L. Stunz, D. A. Mower, R. F. Ashman. 1993. Apoptosis in splenic B lymphocytes: regulation by protein kinase C and IL-4. J. Immunol. 151:2965.[Abstract]
30. Parry, S. L., J. Hasbold, M. Holman, G. G. B. Klaus. 1994. Hypercross-linking surface IgM or IgD receptors on mature B cells induces apoptosis that is reversed by costimulation with IL-4 and anti-CD40. J. Immunol. 152:2821.[Abstract]
31. Zamorano, J., H. Y. Wang, L.-M. Wang, J. H. Pierce, A. D. Keegan. 1996. IL-4 protects cells from apoptosis via the insulin receptor substrate pathway and a second independent signaling pathway. J. Immunol. 157:4926.[Abstract]

This article has been cited by other articles:

				Y. Do, A. Q. Rafi-Janajreh, R. J. Mckallip, P. S. Nagarkatti, and M. Nagarkatti Combined deficiency in CD44 and Fas leads to exacerbation of lymphoproliferative and autoimmune disease Int. Immunol., November 1, 2003; 15(11): 1327 - 1340. [Abstract] [Full Text] [PDF]

				H. He, P. A. Hershberger, and S. A. McCarthy Characterization of a Novel Bax-Associated Protein Expressed in Hemopoietic Tissues and Regulated During Thymocyte Apoptosis J. Immunol., August 1, 1998; 161(3): 1169 - 1175. [Abstract] [Full Text] [PDF]

				H. He, P. A. Hershberger, and S. A. McCarthy Down-Modulation of a Novel Bax-Associated Protein During Apoptosis in Normal Mature B Lymphocytes J. Immunol., August 1, 1998; 161(3): 1176 - 1182. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by He, H. Articles by McCarthy, S. A. Search for Related Content PubMed PubMed Citation Articles by He, H. Articles by McCarthy, S. A.