Difference in apoptosis induction between surface IgD and IgM
David Peckham1,
Erica Andersen-Nissen2,
Fred D. Finkelman3,
Laura L. Stunz1 and
Robert F. Ashman1,2
1 Department of Medicine, Iowa City Veterans Administration Medical Center, Iowa City, IA 52242, USA
2 University of Iowa College of Medicine, Iowa City, IA 52242, USA
3 Department of Medicine, University of Cincinnati College of Medicine and Cincinnati Veterans Administration Medical Center, Cincinnati, OH 45267, USA
Correspondence to:
R. F. Ashman, Department of Medicine/Rheumatology, University of Iowa, C31-P GH, Iowa City, IA 52242 USA
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Abstract
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In the classic `two-signal' model for B cell activation, signal 1 through the antigen receptor plus signal 2 through lymphokine receptors and CD40 leads to proliferation, but signal 1 alone leads to tolerance or anergy. In a protocol designed to deliver signal 1 in vitro with anti-
without signal 2, purified small dense B cells from untreated mice exposed to any of three monoclonal anti-
antibodies or to polyclonal anti-
in vitro showed modest S phase entry at 50 µg/ml. In contrast, at low doses (0.10.5 µg/ml) of anti-
, there was no cell cycle entry at 64 h, but apoptosis was accelerated at 16 h. Polyclonal anti-µ and three monoclonal anti-µs did not show this early apoptosis induction. Anti-CD40 and IL-4 inhibited apoptosis in B cells treated with 0.5 µg/ml anti-
and increased S phase entry at 10 µg/ml anti-
. Low-dose anti-
(but not anti-µ) induced increased B7-2 and class II MHC expression on a subset of B cells, many of which were in apoptosis. Larger transient increases in c-Myc and Egr-1 expression were seen with low-dose anti-
than with anti-µ, followed by an abrupt fall below baseline, a sequence previously linked to apoptosis. There was no change in Bcl-2, Bcl-xL or Bax. These data suggest a functional difference between
and µ cross-linking on resting spleen B cells. A BCR stimulus sufficient for early activation events, but insufficient for full G1 entry, may lead to apoptosis.
Keywords: anti-
, apoptosis, B lymphocytes, cell cycle entry, spleen
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Introduction
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In 1968, Bretcher and Cohn described their `two-signal' model of B cell activation, wherein antigen provides an initiation signal through the BCR (signal 1), and Th cells provide a progression signal (signal 2) via CD40 and lymphokine receptors (1). The currently favored concept is that if a mature B cell receives signal 1 without signal 2, anergy or apoptosis occurs, resulting in immunologic tolerance. Sensitivity to this mode of tolerance induction is greatest in neonatal B cells (2) and the centrocyte B cells of germinal centers (35).
Mystery still surrounds the question of why mature naive B cells express sIgD as well as sIgM. Detailed examination of sIg-initiated signal transduction has failed to reveal important differences between sIgD and sIgM (6,7). Furthermore, mice transgenic for either sIgD anti-hen egg lysozyme (HEL) or sIgM anti-HEL both contain B cells with the ability to respond with activation or tolerance at the appropriate developmental stage (8). Newly developed IgD+IgM+ B cells 14 days after irradiation respond to high concentrations (2550 µg/ml) of either anti-
or anti-µ with apoptosis, whereas mature spleen B cells (which express up to 1 log more sIgD than sIgM) (9) respond to these same concentrations of anti-
or anti-µ with [3H]thymidine incorporation (10). These newer data appear more convincing than older arguments suggesting that IgD may promote immunity over tolerance (11,12).
To study B cell activation and tolerance in vivo, Finkelman et al. injected mice with monoclonal anti-
antibodies (13,14), more recently using a protocol devised to block T cell help, Fc
RII receptor engagement and the generation of new immature B cells from the bone marrow (15). This treatment resulted in the up-regulation of MHC class II on B cells, indicating activation (signal 1), but between day 3 and day 10 most of the B cells disappeared from lymphoid organs (15). Can apoptosis be the fate of mature B cells which receive signal 1 without signal 2, just as it appears to be the fate of immature B cells encountering self-antigens at a time when no T help is available?
The experiments reported here provide direct evidence that mature resting spleen B cells do respond with apoptosis to an isolated signal 1 delivered through surface IgD much more readily than to a signal delivered through surface IgM. Kozono et al. have shown that when B cells of most mouse strains are exposed in vitro for 18 h to anti-µ antibodies the percent of apoptotic cells is 1015% greater than if they are exposed only to medium, but they did not study anti-
antibodies (16). In the current study we demonstrate that polyclonal and monoclonal anti-
antibodies accelerated apoptosis much more effectively than anti-µ antibodies. This effect was seen at concentrations ~1/50 of those necessary to drive cells into G1. In contrast, polyclonal and monoclonal anti-µ antibodies were just as effective as anti-
at driving cells into G1. B cells exposed to apoptosis-accelerating concentrations of anti-
showed signs of early activation, such as increased expression of B7-2, c-Myc and Egr-1, but no change in Bcl-2, Bcl-xL or Bax.
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Methods
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Cell source
Specific pathogen-free BALB/c mice (1016 weeks of age) were purchased from Jackson Laboratory (Bar Harbor, ME) and housed in a specific pathogen-free facility with virus-antibody surveillance. Spleen B cells were isolated at 9597% purity of B220+ cells by panning in 3% BSA (17) followed by treatment with anti-Thy-1.2 (Accurate Scientific, Westbury, NY) plus complement. Percoll gradient centrifugation yielded a small dense G0 phase B cell population at the 49%/76% interface (
= 1.0611.098).
Cell culture
B cells were cultured in complete RPMI 1640 medium (Gibco/BRL, Grand Island, NY), supplemented with 5% FCS, 1 mM sodium pyruvate, 10 mM HEPES, 0.1 mM non-essential amino acids and 1 mM L-glutamine. They were cultured in 96-well Costar plates in 200 µl of medium at a concentration of 2x106 cells/ml in a water-saturated atmosphere containing 5% CO2 at 37°C.
Reagents
Rat IgG1 anti-mouse Cµ2 mAb B.7.6 (18) was a gift from Thomas Waldschmidt. Polyclonal goat IgG anti-mouse µ utilized in flow cytometry experiments was from Jackson Laboratories (West Grove, PA). H
a/1 mouse IgG2b anti-
a (19), HB
7 rat IgG2a anti-
(officially called LO-MD-7), 11-26 (a poorly cross-linking rat IgG 2a anti-
-15), HB
6 rat IgG2a anti-
(officially called LO-MD-6), GK1.5 rat IgG2b anti-CD4, m25 rat IgG1 anti-human/mouse IL-7 (19) and 2.4G2 rat IgG2b anti-mouse Fc
RII (20) were grown as ascites, and purified by (NH4)2SO4 precipitation and DEAEcellulose ion exchange. The m25 was grown in BALB/c mice, the H
a/1 in BALB/c xC57BL/6 F1 mice, and the GK1.5, 11-26 and 2.4G2 in athymic nude mice. LO-MD-6 and LO-MD-7 were a gift of Dr Hervé Bazin. Anti-CD40 was from PharMingen (La Jolla, CA). B220Cy5 was a gift of Dr John Cowdery (University of Iowa). IL-4 (Genzyme, Cambridge, MA) was used at 25 U/ml, a concentration which inhibits apoptosis in B cells (21). Monoclonal anti-µ antibodies DS-1 (22) and 331 were derived from ascites from plasmacytoma-bearing mice.
Flow cytometric measurement of apoptosis and cell cycle entry
Because the fluorescent dye acridine orange (AO; Polysciences, Warrington, PA) binds RNA and DNA meta-chromatically, the percent of cells in G0, G1, S/G2/M and apoptosis can be determined (23). First, B cells were permeabilized in a buffer containing 0.1% sodium citrate, 0.02 M phosphate, 0.1% Triton X-100, 0.2 M sucrose and 100 µM disodium EDTA, pH 3.5. One minute later, 20 µg/ml AO in 0.01 M phosphate and 0.1 M NaCl, pH 3.8, was added. Cells were analyzed on a Becton Dickinson FACS scan, using a 488 nm argon laser excitatory band, a 525 nm band-pass filter for DNA and a 635 nm band-pass filter for RNA. Boundaries between G0 and apoptosis and between G0 and G1 were determined on DNA versus RNA histograms of fresh cells and cells that had been cultured 16 h in medium alone, which contained a discrete population of apoptotic cells. Hypodiploid cells had less DNA than G0 cells. Debris cut-offs were set at 25% of G0 DNA and the left boundary of G0 for RNA.
Lymphocytes were assessed for B220FITC binding and DNA cleavage (percent hypodiploid nuclei). Cells were stained with B220FITC in PBS + 0.5% BSA at 4°C. They were then washed in PBS before fixation in 70% EtOH at 4°C. Cells were washed and resuspended in PBS containing RNase (250 µg/ml) and propidium iodide (50 µg/ml). Samples were incubated for 1 h at 37°C before flow cytometric analysis on an Epics 753 flow cytometer (Coulter, Hialeah, FL) using a 488 nm argon laser excitatory band, a long-pass 550 nm dichroic mirror, 525 and 630 nm band-pass filters, and a short-pass 600 nm dichroic mirror.
In one set of experiments, purified B cells were exposed first to 5 µg/ml anti-B220 conjugated to Cy5, then sorted on the Epics 753. Part of the sorted population was recombined with non-B cells to control for sorting artifacts. In another experiment, to assess viable cell recovery, cell suspensions were spiked at the time of analysis with equal volumes of a suspension of 2.05 µm beads (Coulter). These beads had forward and side scatter which was uniform and easily distinguished from cells. Cell recovery was then measured by flow cytometry as the number of particles falling into the live plus apoptotic cell gates per 10,000 beads counted in the bead gate. In this experiment the cells were assayed for MC540 binding as described in (26), which correlates completely with Annexin-V binding in our hands, staining both apoptotic and membrane-permeable cells. In contrast, erythrosin B distinguishes membrane-permeable cells only.
To assess whether apoptosis was occurring in B cells which were partially activated, B cells were prepared from BALB/c mice by the standard protocol and cultured in medium alone or with 25 U/ml IL-4, 0.5 µg/ml anti-
(H
a/l) or 0.5 µg/ml anti-µ (B.7.6) for 18 h. The cells were treated with 2.4G2 (Fc block; PharMingen) to block Fc
RII receptors, then directly stained with either FITC-conjugated rat anti-mouse CD86 (B7-2) (PharMingen) or with FITC-conjugated anti-Iad (M
D6). FITC-conjugated mouse IgG1 and FITC-conjugated rat IgG were used as staining controls. After washing, the cells were stained with Annexin Vphycoerythrin (PharMingen) in Annexin V-binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) at 4°C for 15 min. Cells were then suspended in a larger volume of binding buffer and analyzed on the Epics 753 (Coulter) using a 488 nm argon laser excitatory band, 525 and 670 nm band-pass filters, and a DM570 dichroic mirror.
Western blots
Cell (16x106/sample) were cultured as described (24), harvested, washed twice in PBS and lysed in lysis buffer (1.2% NP-40, 150 mM NaCl, 25 mM HEPES, 5 mM NaF, 0.5% sodium deoxycholate supplemented with 1 mM PMSF, 10 mM Na3VO4, 4.4 mg/ml ß glycerophosphate, 10 mM sodium pyrophosphate, 5 µg/ml each of antipain, aprotinin, leupeptin and trypsin inhibitor, 0.5 µg/ml pepstatin, 7.5 µg/ml bestatin, and 4 µg/ml phosphoramidon; Boehringer-Mannheim). After 30 min on ice, the lysates were centrifuged 15 min at 12,000 g, 4°C. The supernatants were transferred to new tubes and stored at 20°C. Protein was quantitated using the BioRad assay. Aliquots of 40 µg protein per lane were loaded on 10, 12 or 15% Trisglycine gels and electrophoresed under denaturing conditions. The proteins were electroblotted onto Immobilon P membranes (Millipore, Bedford, MA). Membranes were blocked for 2 h in 5% non-fat milk in TBS + 0.5% Tween 20 (24). The blots were incubated in TBS with 0.5% Tween containing the following rabbit IgG primary antibodies: anti-Egr-1, anti-Bcl-xS/L, anti-c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA); anti-Bcl-2 (Calbiochem, San Diego, CA); and anti-c-Myc (Upstate Biotechnology, Lake Placid, NY). Blots were also probed with rat IgM anti-Bax (PharMingen). Secondary antibodies used were donkey anti-rabbit IgGhorseradish peroxidase (Amersham, Arlington Heights, IL) and goat anti-rat IgMhorseradish peroxidase (Cappel, Durham, NC). Blots were developed with ECL reagents (Amersham) and exposed to film.
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Results
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Anti-
is more effective than anti-µ at accelerating apoptosis in B cells at low concentrations in vitro
In short-term cultures of purified small dense spleen B cells we used AO staining (23) in order to derive the percent of apoptotic cells as well as all the phases of the cell cycle from the same histograms. At concentrations 10- to 100-fold too low for cell cycle entry, anti-
induced more apoptosis in vitro than anti-µ (Figs 15



). For example, at 0.11 µg/ml anti-
H
a/1, accelerated apoptosis occurred at the expense of the G0 population at 16 h (Fig. 1A
). No entry from G0 into G1 was observed at 64 h at anti-
concentrations of
10 µg/ml (Fig. 1B
). By 64 h, at 10100 µg/ml of H
a/1, about half the cells were in G1 or S phase and the percent of apoptotic cells was less than that seen with 01 µg/ml. These results were obtained with resting B cells under conditions of extreme T cell depletion; however, to eliminate the possibility that the effect of high-dose anti-
on B cell cycle entry required another cell, in one experiment we compared the H
a/1 doseresponse curves (0.150 µg/ml) of sort-purified B220+ cells to sorted, recombined cells and unsorted cells, measuring cell cycle entry at 64 h, and percent apoptotic B220+ cells, finding no difference (data not shown). IL-4 reduced the in vitro apoptosis rate of B cells treated with 0.5 µg/ml anti-
H
a/1 to the rate seen in medium alone at 16 h (Table 1
). By 64 h this protection was lost (not shown). Anti-CD40, especially in combination with IL-4, also inhibited anti-
-induced apoptosis and caused cell cycle entry by 64 h (Table 1
).

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Fig. 4. BALB/c B cells were incubated with a range of concentrations of monoclonal anti-µ antibodies DS-1 or 331, then assayed at 16 and 64 h by AO flow cytometry to determine cell cycle status. Apoptotic () and cycling (G1M) ( ) are shown. Means ± SE of three independent experiments. The mean percent of apoptotic cells in unstimulated cultures was 46.9 ± 11% at 16 h and 86.3 ± 5.6% at 64 h. The mean number of cycling cells in unstimulated cultures was 2.6 ± 1% at 16 h and 3.7 ± 2.5% at 64 h. Closed triangles show % apoptotic cells seen with 0.5 µg/ml Sa/1 in the same experiments.
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Since we used whole anti-
, the possibility of Fc receptor interaction must be considered. However, anti-Fc
RII antibody 2.4G2 failed to block apoptosis acceleration by 0.1 µg/ml H
a/1 (Fig. 1A and B
). The generalizability of our results to other anti-
antibodies was tested in the experiment shown in Fig. 2
. Monoclonal HB
7 and polyclonal goat anti-mouse-
both accelerated apoptosis like H
a/1 at 16 h (Fig. 2A
) and drove B cells into cycle by 64 h (Fig. 2B
). Antibody 11-26, which cross-links poorly (15), was much less effective at induction of both apoptosis and cell cycle entry. Apoptosis induction required a 10-fold higher concentration of 11-26 than of other anti-
s. In contrast, anti-µ antibody did not show the early apoptosis-accelerating effect of anti-
. B.7.6 anti-µ, frequently used for its superior stimulatory properties, showed only a small increase in apoptosis at 0.11 µg/ml, both at 16 and 64 h (Fig. 3
). Two less stimulatory monoclonal anti-µs, DS-1 and 331, showed even less increase in apoptosis (Fig. 4
). Polyclonal anti-µ showed a small increase similar to that of B.7.6 (Fig. 5
). As with anti-
, G1 entry with B.7.6 or polyclonal anti-µ required at least 10 µg/ml (Figs 1 and 3
).
A useful way to contrast the anti-
and anti-µ results is to consider the difference in percent of apoptotic cells between exposure to medium alone for 16 h and exposure to 0.51 µg/ml antibody. For anti-
H
a/1, this difference was 32% in Fig. 1
, whereas in Fig. 2
it was 34% for H
a/1, 37% for HB
7, 32% for polyclonal anti-
(all of which induced substantial G1 entry at 50 µg/ml) and 27% for 11-26 (which did not induce G1 entry). In contrast, for the anti-µs this difference was 11% for B.7.6 (Fig. 3
), 15% for DS, 11% for 331 (Fig. 4
) and 9% for polyclonal anti-µ (Fig. 5
). After 64 h exposure to 1050 µg/ml of either anti-
or anti-µ the percentage of apoptotic cells was often below the level observed in medium alone (Figs 1 and 36



) whether substantial cell cycle entry was evident (Figs 1, 3, 5 and 6


) or not (Fig. 4
).
Seeking an alternative assay for early apoptosis, we performed doseresponse curves with B.7.6 and polyclonal anti-µ using MC540 to detect the loss of membrane phospholipid asymmetry at 16 h (25, 26). The results resembled the AO data of Figs 35

: 3941% MC540+ cells at 00.01 µg/ml, rising only to 4850% at 1 µg/ml. In one 16 h experiment, we observed 3% B cell apoptosis by AO in fresh cells, 29% in medium, 47% in 0.1 µg/ml anti-
(H
a/1) and 48% in 0.1 µg/ml anti-
plus 0.1 µg/ml anti-µ (B.7.6), indicating that anti-µ did not interfere with the apoptosis-inducing activity of anti-
.
Three experiments were performed to verify that the observed apoptosis progressed to loss of viable cells. In the first, erythrosin B was used as an indicator for cells with damaged plasma membranes after duplicate samples of 2x106 cells were cultured for 16 h with 0.05, 0.1, 0.5 or 1 µg/ml H
B7 anti-
antibody (the apoptosis inducing range in Fig. 2
), 25 µg/ml lipopolysaccharide or medium. Erythrosin B cell recoveries in the samples in medium alone were 770 and 730x103, and in lipopolysaccharide 820 and 770x103 (16 h is too early for proliferation). Each of these counts was >2 SD higher than the mean of 485x103 ± 111 (SD) seen in the eight anti-
cultures. The second and third contrasted cell recoveries with 0.5 µg/ml anti-
or anti-µ (Table 2
), using two alternative viability measures, MC540 at 20 h (which stains apoptotic and membrane-permeable cells) in experiment 1 and erythrosin B at 40 h (which stains only membrane-permeable cells) in experiment 2. In both these experiments viable cell recovery was less with the anti-
antibodies than with anti-µ antibodies and greater without any antibodies. However, total cell recovery was not consistently different.
The discovery by Hasbold and Klaus that hyper-cross-linking of surface Ig leads to apoptosis (27) was prompted by a previous demonstration that plates incubated with 50100 µg/ml anti-
and anti-µ caused apoptosis, whereas soluble antibody in the same concentration range led to proliferation (28). Figure 6
compares the doseresponse curves for the plate-bound and soluble forms of H
a/1 within the same experiment, and the result was entirely consistent with the data of Hasbold and Klaus. At both 16 and 64 h, plates incubated with 50 µg/ml H
a/1 stimulated increased apoptosis, with no progression into cycle; by 64 h, 10 µg/ml was also effective. However, this effect was duplicated by about 1/200 as much anti-
in solution, which did stimulate cell cycle entry at 1050 µg/ml, as in Fig. 1
.
Insight into the mechanism of the apoptosis-accelerating effect of anti-
was sought by Western blot analysis of selected cellular oncogenes and Bcl-2 family members in B cells treated with a dose (0.2 µg/ml) of anti-
sufficient to cause apoptosis but not cell cycle entry (Fig. 1
) or a matching dose of anti-µ, using a stimulating anti-CD40 antibody as a positive control. Neither anti-
nor anti-µ altered levels of Bcl-2, Bcl-xL or Bax (Fig. 7
). Anti-
H
a/1 induced an early burst of Egr-1 and c-Myc protein, visible at 3.5 h and gone by 7 h, which was greater than the otherwise similar changes seen with monoclonal anti-µ (Fig. 7
). Anti-CD40, which is apoptosis protective (29), induced a more lasting increase in c-Myc and also increased Bcl-xL (Fig. 7
). Fig. 7
emphasizes that the levels of c-Myc and Egr-1 are below baseline by 7 h. They stay low at least to 16 h when they differ little from levels seen with medium alone (not shown). There was no detectable change in c-Jun or c-Fos levels with low-dose anti-
or anti-µ (not shown).
B7-2 and class II MHC are up-regulated early in B cell activation (30). The low concentration of H
a/1 anti-
that induced apoptosis without cell cycle entry in Fig. 1
was able to increase both B7-2 and class II MHC (Fig. 8A and B
). An equal concentration of B.7.6 anti-µ did not increase B7-2 or class II MHC (Fig. 8A and B
). H
a/1-treated B cells displaying high B7-2 and class II MHC appeared in both apoptotic (Annexin V+) and non-apoptotic populations. A small population of apoptotic cells (Annexinhigh) bound more anti-B7-2 than any of the live (Annexinlow) cells (Fig. 8D
). No similar high-binding population appeared on the anti-class II MHC histograms (Fig. 8E
). As a further control, cells exposed to IL-4 (the best inducer of B7-2 and class II MHC in this experiment) and stained with FITC-conjugated non-specific Ig, showed no excess uptake of Ig by Annexinhigh cells and much lower fluorescence intensity than either FITCanti-B7-2 or FITCanti-Ia (Fig. 8C
). Thus the population in the box in Fig. 8
(D) represents selective high expression of B7-2 by a population of apoptotic cells. This population varied as a function of the percentage of apoptotic cells in every condition tested [compare the 11.5% seen with 0.5 µg/ml anti-
in Fig. 8
(D) to 1.3% in fresh cells, 3.5% in medium alone, 2.7% in IL-4, 7.6% in 0.5 µg/ml B.7.6 anti-µ and 26.6% in 10 µg/ml cycloheximide a potent acceleration of apoptosis] (21). In summary, low-dose anti-
, but not anti-µ, increases the expression of I-A and B7-2, and this change remains evident even after cells enter apoptosis. However, in addition, a population of apoptotic cells binds high amounts of anti-B7-2 (not anti-I-A) regardless of the means of apoptosis induction.
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Discussion
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This study provides evidence that the direct interaction between B cells and anti-
antibody may induce early (16 h) apoptosis at low concentration (0.11 µg/ml) or cycle entry at high concentration (10100 µg/ml, Fig. 1
). Apoptosis acceleration was much less evident with several anti-µ antibodies (Figs 35

) than with anti-
antibodies (Figs 1, 2 and 6

). This difference was seen with both monoclonal and polyclonal anti-
and anti-µ antibodies (Fig. 2
). Several explanations for the difference in the apoptosis-inducing effects of anti-µ and anti-
may be considered. The small dense B cell fraction (called `resting' because it shows baseline expression of several activation markers and no spontaneous progress out of G0) expresses much more IgD than IgM (9). For this reason alone, IgD cross-linking could generate more clustered Ig
and ß than surface IgM cross-linking. Furthermore, different antibodies bind to surface Ig with different affinities and geometries, which could affect their signaling properties. We considered how to adjust conditions so that anti-
and anti-µ were delivering an `equivalent signal', but none of the proximal signaling events was free of alternative interpretations. We decided on the strategy of using multiple mAb (three anti-
s and three anti-µs) over a 4-log concentration range to convince ourselves that we were dealing with a consistent isotype-based difference. At no concentration did any of the three monoclonal anti-µs or the polyclonal anti-µ achieve the degree of apoptosis acceleration seen with any of the three monoclonal anti-
s or the polyclonal anti-
(Figs 16




). After 16 h exposure to 0.51 kg/ml of four different anti-
s, the increase in percent apoptotic cells ranged from 27 to 37%, whereas with four different anti-µs, the difference ranged from 9 to 15% (Figs 15



). The anti-
believed to have the lowest cross-linking potential (11-26) required 10 times more than the other anti-
s to accelerate apoptosis (Fig. 2
). We concluded that on resting spleen B cells, low-level cross-linking of surface IgD and IgM differ in their effects on apoptosis, whereas a higher level of cross-linking of either isotype induces similar entry into G1.
Cell cycle entry at 10100 µg/ml anti-
did not require contaminating T cells because sort-purified B220+ cells had a similar anti-
dose response (data not shown). Monoclonal and polyclonal anti-µ antibody at 10100 µg/ml also stimulated cell cycle entry by 64 h. With 0.11 µg/ml B.7.6 and polyclonal anti-µ, there was a modest increase in apoptosis at 64 h (Fig. 3
) which was not seen with 331 or DS-1 (Fig. 4
). However, the striking early (16 h) increase seen with 0.11 µg/ml anti-
was not observed with anti-µ, either as hypodiploid cells (Figs 35

) or the plasma membrane transition detected with MC540 (see Results). In the animal, such a delay would give the anti-µ-treated B cell a greater opportunity to encounter signal 2 and thereby escape death. Since 0.1 µg/ml anti-µ did not interfere with the apoptosis-inducing effect of 0.1 µg/ml anti-
, we concluded that in resting mature B cells anti-µ provides a much weaker signal for early apoptosis than anti-
, rather than blocking such a signal.
The modest apoptosis-accelerating effect of anti-µ B.7.6 (Fig. 3
) and polyclonal anti-µ (Fig. 5
) in BALB/c B cells was consistent with that reported by Kozono et al. (16). In our data, 16 h of exposure to 0.51 µg/ml B.7.6 (Fig. 3
) or polyclonal anti-µ (Fig. 5
) increased the percent of apoptotic cells from 36 to 4850%. In the data of Kozono et al., 1 µg/ml polyclonal goat anti-µ increased the percent apoptotic cells from 41 to 56% at 18 h in BALB/c B cells. Other normal strains showed apoptosis percentages 1219% greater in anti-µ than in medium at 18 h, whereas NZB B cells showed a smaller difference (~5%). Kozono et al. did not test anti-
antibodies (16), nor did they test DS-1 or 331, anti-µ antibodies that failed to accelerate apoptosis at any concentration in our hands (Fig. 4
).
B cells responding to 0.11 µg/ml anti-
appeared to go into apoptosis directly from G0, similar to B cells in medium alone (Fig. 1
); indeed without anti-CD40 or IL-4, these B cells continue to progress into apoptosis without much G1 entry between 16 and 64 h. At 10100 µg/ml, the 25:75% split between apoptosis and G0 at 16 h resembled that seen in medium alone. With no further increase in apoptotic cells, between 16 and 64 h the increase in cycling cells was balanced by a decrease in G0 cells (Fig. 1
). Thus, B cells responded to low concentrations of anti-
with apoptosis and no cycle entry throughout their course, whereas at high concentrations cycle entry was associated with apoptosis protection (i.e. a rate of apoptosis lower than the spontaneous rate) even in the absence of `signal 2'. B cells exposed to signal 1 in the form of 0.11 µg/ml anti-
were rescued from early apoptosis by signal 2 in the form of anti-CD40 (even at the suboptimal level of 66 ng/ml) and IL-4, so that more cells survived to enter cycle by 64 h (Table 1
). In experiments not shown, 25 µg/ml anti-CD40 alone kept apoptosis levels at baseline in B cell cultures for at least 64 h.
Cross-linking surface Ig to Fc
RII on B cells has been shown to accelerate apoptosis, presumably contributing to the inhibition of cell cycle entry (31). This effect is blocked by 25 µg/ml 2.4G2 anti-Fc
RII antibody. Inclusion of 25 µg/ml 2.4G2 with 0.1 µg/ml anti-
did not curtail apoptosis in vitro (Fig. 1A
) nor did 2.4G2 administration prevent anti-
-induced apoptosis in vivo (32), arguing against an important role for Fc
RII in anti-
-induced apoptosis.
In a few experiments we monitored cell recovery, with other measures for determining viability (Table 2
). Anti-
antibodies always yielded fewer viable cells than anti-µ antibodies, at several time points, but even anti-µ antibodies appeared to increase cell death. However, in the time frame studied, the total (live and dead) cells recovered was not decreased by these antibodies.
Because polyclonal antibody can engage more epitopes per surface Ig molecule than mAb, especially at supersaturating concentrations like 25100 µg/ml, one might expect to see an effect of Ig cluster size in this experiment. However, monoclonal anti-
antibodies were at least as adept at inducing apoptosis as polyclonal antibody (Fig. 2
), suggesting that linear aggregates of surface Ig were sufficient for the apoptosis signal. Klaus et al. observed that when plastic plates were incubated with anti-µ or anti-
, washed and B cells were added, apoptosis was greater than when B cells were exposed directly to the same 50 µg/ml concentration of antibody (28). They concluded that plate-bound antibody produced apoptosis because it effected a higher degree of cross-linking. Our doseresponse data is consistent with theirs, but suggests that at least for anti-
a different explanation should be considered: that B cells on the plastic plate may be exposed to only ~0.5% as much undenatured anti-
as B cells incubated with soluble antibody (Fig. 6
). There is evidence that much plate-bound antibody is inactivated, but that elution of intact antibody is in the 12% range (J. E. Butler, pers. commun.).
Another variable affecting how B cells respond to surface Ig engagement is the extent of cross-linking. Hasbold and Klaus have shown evidence that whereas both anti-
and anti-µ at 50 µg/ml can activate (again consistent with Figs 15



), hyper-cross-linking of biotinylated anti-
or anti-µ with avidin leads to apoptosis (27). Our data completes this picture by suggesting that degrees of cross-linking less than the optimum for activation can also induce apoptosis in mature B cells, but that in this low dose range, surface IgD transmits early apoptosis signals more readily than IgM.
The concept that surface IgD may be more likely to send an apoptosis signal than IgM is at variance with much of the previous literature. IgD expression is delayed until B cells are mature enough to migrate to the periphery. Whereas mature B cells express IgD as well as IgM and respond to antigen with activation, neonatal B cells lack surface IgD and respond to the same concentration of antigen with tolerance (2). These results suggested that engagement of surface IgD might block tolerance induction. The WEHI 231 cell line, with its immature M+D phenotype, has been extensively studied because anti-µ triggers apoptosis (33,34). When IgD heavy and light chain-encoding vectors were transfected into WEHI 231 and expressed, anti-
would still not cause apoptosis (3436). These results suggested engagement of surface IgM alone might favor tolerance induction. These conclusions were supported by experiments in which the sensitivity of spleen B cells to tolerance induction in vitro was increased by prior removal of surface IgD (37). Furthermore, in IgM anti-TNP transgenic mice, in vivo treatment with anti-µ or multivalent TNP-antigens led to selective deletion of IgMbright cells in the spleen (11), resembling that seen in normal mice with anti-
(15). When surface IgD anti-TNP was co-expressed with IgM, deletion with antigen was much less effective. Apoptosis was detected by DNA ladder in fresh IgM transgenic cells from mice treated with antigen 24 h before, but there was no comparison made to cells from IgM + IgD transgenics (11). Thus it seems likely that conditions exist wherein IgM can also send an apoptosis-predominant signal in vivo or in vitro when engaged by plate-bound anti-µ (28), even though it was difficult to detect in vitro with soluble anti-µ antibody (Figs 4 and 5
).
Studies of signal transduction via IgM and IgD have so far failed to find differences between their downstream effects (6,7). For example, Norvell and Monroe found that mature M+D+ spleen B cells responded with thymidine incorporation to both anti-
and anti-µ, whereas immature M+Dlow B cells responded to neither (10). The doses of anti-µ and anti-
used were 25 and 50 µg/ml, consistent with our results in the high dose range (Figs 13 and 5


). Lower concentrations were not tested.
It is impossible to compare the effects of in vitro to in vivo administration of anti-
because the concentration and duration of exposure experienced by B cells after a parenteral injection of anti-
would be impossible to determine or duplicate in vitro. Nevertheless, it is intriguing that 100 mg HB
6 injected i.p. induces loss of 90% of spleen B cells by day 10 under conditions where pretreatment with GK1.5 anti-CD4 has prevented T help, anti-IL-7 has prevented recruitment of new B cells from the marrow and 2.4G2 anti-Fc
RII has prevented B cell opsonization (15). We reproduced these experimental conditions, and showed that on days 1, 3, 5 and 10 after anti-
administration, B220+ spleen cells (but not B220 cells) showed accelerated spontaneous apoptosis rates in 16 h cultures, providing specific evidence that the anti-
-induced disappearance of B cells was due to apoptosis (32). If T help is present, anti-
in vivo activates B cells instead (15) just as anti-CD40 and IL-4 rescued B cells in vitro from anti-
-induced apoptosis (Table 1
).
B cells of mice receiving anti-
in vivo have been shown to display increased class II MHC expression as a sign of early activation (15), even as they are disappearing because of apoptosis. Similarly, many of the B cells exposed in vitro to 0.2 µg/ml of anti-
expressed elevated levels of B7-2 and Ia (Fig. 8A and B
), even though they were not destined to attain G1 (Fig. 1
). Indeed, some of the cells expressing these early signs of activation also expressed evidence of apoptosis in the form of increased Annexin V binding (Fig. 8D and E
) and loss of mitochondrial membrane potential (not shown), characteristics of early apoptosis (26,38). Thus early activation and apoptosis events can occur in the same cells, when they receive a modest BCR signal without a co-stimulatory signal.
Seeking a molecular explanation for the difference in the early apoptosis-promoting effect of low-dose anti-
and anti-µ, we tested their effect on cellular oncogene and apoptosis regulator expression. Although there is an association between increased c-Myc and increased apoptosis in fibroblasts (39), c-Myc and Egr-1 are generally associated with cell cycle progression in lymphocytes. Rapid loss of c-Myc is associated with apoptosis in WEHI-231 B cells (4042) and also in normal spleen B cells (43,44), especially if the fall is preceded by an abrupt rise (41,42). So it may be highly pertinent to apoptosis acceleration that low-dose anti-
induced a higher earlier rise in the level of oncogene products Egr-1 and c-Myc than either anti-µ preparation, which accentuated the subsequent rapid drop to below baseline levels seen with both kinds of antibody (Fig. 7
). Alternatively, elevated c-Myc and Egr-1 might lead to apoptosis if other proteins that are only induced by high concentrations of anti-
or anti-µ are absent, in which case the magnitude of the rise would be the deciding factor. We observed no change in c-Jun (not shown) nor in Bcl-2, Bcl-xL or Bax with low-dose anti-
or anti-µ (Fig. 7
). With high-dose anti-
(10 µg/ml H
a/1), RNase protection experiments by D. Peckham (unpublished) have shown the elevated level of c-myc mRNA to remain well above baseline for 16 h.
Our results suggest that by sending an apoptosis signal under conditions of low-level cross-linking, surface IgD may play a greater role in tolerance induction of mature spleen B cells than previously supposed. That simultaneous engagement of IgM and IgD had the same apoptosis-inducing effect as engagement of IgD would predict that antigen might have a similar effect. There may be a low antigen concentration range which sends an apoptosis signal through IgD and little or no signal through IgM (Figs 1, 4 and 5

), especially in B cells expressing more D than M. When antigen is concentrated, as in a lymphoid follicle, B cells encountering high-intensity signal 1 (simulating
10 µg/ml in Figs 13

) may be less dependent on signal 2 from T cells to be stimulated into cycle. In Table 1
, at 64 h, addition of IL-4, anti-CD40 or both to 10 µg/ml H
a/1 only raised the percent in cycle slightly, whereas they made a much greater difference with 0.5 µg/ml H
a/1. Further from the follicle center, where antigen concentration is lower, the fate of the B cell hangs in the balance between cycle entry when signal 2 is available (45) and apoptosis when signal 2 is absent (Fig. 1
and Table 1
).
In summary, this paper provides an especially clear illustration of the principle that a BCR signal without a co-stimulatory signal 2 leads to apoptosis. The anti-
-induced disappearance of B cells in CD4+ cell-depleted mice in vivo is directly demonstrated to be due to apoptosis, as shown most clearly by an accelerated ex vivo apoptosis rate. In vitro, concentrations of anti-
too low to drive G1 entry, but sufficient to trigger early activation events, caused early apoptosis acceleration which was prevented by `signal 2' from IL-4 or anti-CD40. Neither polyclonal nor monoclonal anti-µ could quantitatively match these effects of anti-
. In the past, consistent functional differences between surface IgD and IgM have been elusive to say the least, but apoptosis induction is an intriguing candidate for further investigation.
 |
Note
|
---|
Figure 1
has been previously presented in a published meeting symposium (32). When E. A.-N. presented Figs 3 and 5
at a conference for research by Minnesota undergraduates, she was invited to publish her presentation in the Minnesota Academy of Sciences Journal.
 |
Acknowledgments
|
---|
We thank Justin Fishbaugh of the University of Iowa Flow Cytometry Facility for technical assistance and Jill Kinnaird for her expert manuscript preparation. We are also grateful to the University of Iowa Microbiology Summer Research Program for support of E. A. N. This research was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs including VA Merit Awards to R. F. A., L. L. S. and F. D. F., a University of Iowa Microbiology Summer Undergraduate Research Fellowship for E. A.-N., and an Arthritis Foundation Biomedical Science Award to F. D. F.
 |
Abbreviations
|
---|
AO acridine orange |
HEL hen egg lysozyme |
 |
Notes
|
---|
Transmitting editor: J. Banchereau
Received 3 April 2000,
accepted 21 November 2000.
 |
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