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*
Medical Services, Department of Veterans Affairs, Iowa City, IA 52246; and
Department of Internal Medicine (Rheumatology), University of Iowa College of Medicine, Iowa City, IA 52242
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous studies have demonstrated that unmethylated CpG dinucleotides,
in particular base contexts (CpG motif) present in bacterial DNA (CpG
DNA) and in certain synthetic oligodeoxynucleotides (CpG ODN), promote
B cell proliferation; secretion of various cytokines, such as IL-6,
IL-12, and IFN-
; and subsequent Ig secretion (10, 11, 12, 13). CpG DNA
synergizes with Ag receptor-mediated signals to increase IL-6 and Ig
secretion and cell proliferation (10, 13). These observations suggest
that the CpG motif could be a costimulatory factor, as well as a
mitogenic factor, for B cell activation.
Like CD40 ligand and LPS, CpG DNA rescues WEHI-231 cells from growth arrest and apoptosis induced by surface Ag receptor cross-linking (14, 15, 16, 17). Because the effects of signaling molecules depend on the cell type and developmental stage of the responding cells and on other environmental factors, the responses of WEHI-231 cells may not mimic those of mature peripheral B cells. Also, continuously cycling cells are inappropriate for investigating the relationship between oligodeoxynucleotide (ODN)3-induced cell cycle entry and apoptosis. Therefore, we investigated whether CpG DNA can provide a survival signal as well as a mitogenic signal for spleen mature B cells in vitro and whether this is associated with changes in the expression of candidate cell proliferation and/or apoptosis-regulating genes. Our results demonstrate that single base changes in ODN produced profound changes in activity that were identical for apoptosis protection and cell cycle entry, that narrow monotonic dose-response curves suggest a uniform population of ODN recognition sites, and that CpG ODNs up-regulate several genes, including c-myc and bclXL. However, cell cycle entry and apoptosis protection could be dissociated when protein synthesis was inhibited.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Escherichia coli (strain B) DNA and calf thymus DNA were purchased from Sigma (St. Louis, MO). Phosphodiester oligodeoxynucleotides (O-ODN) were purchased from Operon Technologies (Alameda, CA). Some were synthesized with a 5-methylcytosine at designated positions. Nuclease-resistant phosphorothioate oligodeoxynucleotides (S-ODN) were purchased from Oligos Etc. (Wilsonville, OR). All DNA and ODN were purified by extraction with phenol-chloroform-isoamyl alcohol (25:24:1) and/or ethanol precipitation. E. coli and calf thymus DNA were made single stranded before use by boiling for 10 min, followed by cooling on ice for 5 min. The LPS level in DNA and ODN was less than 1.7 ng/mg of DNA by Limulus Amebocyte Lysate QCL-1000 (BioWhittaker, Walkersville, MD) following the manufacturer’s protocol.
Mice and cell preparation
B6D2F1 and DBA/2-specific pathogen-free mice at 9 to 18 wk of age were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained under specific-pathogen-free conditions in the University of Iowa animal care unit. Mice were euthanized by cervical dislocation, and their spleens removed using an aseptic technique. Splenic B lymphocytes were obtained by the BSA panning method, described previously (5). Cells obtained in this manner were about 97% surface Ig+B220+.
Culture conditions and reagents
Spleen B lymphocytes were either assayed directly ("0-h" samples) or placed in culture in RPMI 1640 supplemented with 5% (v/v) heat-inactivated FCS, 1 mM L-glutamine, 1 µM sodium pyruvate, 0.1 µM essential amino acids, 10 µM HEPES (Life Technologies, Gaithersburg, MD), and 60 µM 2-ME. Cells (2 x 106/ml) were treated with or without ODN and/or cycloheximide (CHX) and were cultured for 12 to 40 h in 24- or 96-well culture plates (Costar, Cambridge, MA) at 37°C in a 5% CO2 humidified incubator. CHX (Calbiochem, La Jolla, CA) was used at 3 or 10 µg/ml. O-ODN and S-ODN were used at 120 µg/ml and 1 µM (6 µg/ml), respectively, unless otherwise indicated.
FACS analysis of merocyanine (MC)540 and propidium iodide (PI) staining for membrane transition and hypodiploid nuclei
PI was used in separate assays both to quantitate the percentage of hypodiploid nuclei (apoptotic nuclei) and the percentage of whole cells with PI-permeable plasma membranes. MC540 binds strongly to membranes that have lost phospholipid asymmetry, a transition that precedes the cleavage of DNA in apoptotic B cells (7). MC540 binding and PI permeability were measured simultaneously in the same cell suspension. PI staining for both hypodiploid nuclei and membrane permeability, and MC540 assay were done as previously described (7). Whole cells and nuclei were analyzed by a Coulter EPICS 753 Flow Cytometer (Coulter, Hialeah, FL). The lower gates were set to exclude PI-staining fragments with less than 3% of the intensity of a normal nucleus.
Simultaneous analysis of cell cycle and apoptosis by acridine orange (AO)
Cell cycle and apoptosis were analyzed simultaneously by AO staining as previously described (8). AO and permeabilizing buffer were added directly to 2 x 105 harvested, uncentrifuged B cells, and the analysis performed immediately on the FACScan. By staining RNA and DNA different colors, AO provides the distinction between G0 and G1 and enables apoptosis and phases of the cell cycle to be read from histograms.
Preparation of RNA and multiprobe RNase protection assay (RPA)
Freshly isolated spleen B cells (2 x 106 cells/ml) were treated with or without CpG or non-CpG S-ODN (1 µM). Cells were harvested at various time points (15 min to 4 h) and total RNA was isolated by using RNAzol B (Tel-Test, Friendswood, TX) following the manufacturer’s protocol. Gene transcripts were detected using the RPA as previously described (18). Each sample contained 3 µg of RNA before digestion, but to assess loading variation between lanes, we probed for L32, which encodes an ubiquitously expressed ribosome subunit protein (19). The GenBank Accession No. and nucleotide sequence for each gene have been previously described (17).
Preparation of cell extracts and Western blot analysis
Spleen B cells (2 x 106 cells/ml) were treated with medium, CpG, or non-CpG S-ODN (1 µM). Cells were harvested at various time periods (1 to 16 h) after DNA treatment. Cell lysates were prepared as described (17). Equal concentrations of cell lysates were subjected to electrophoresis on a 12% polyacrylamide gel containing 0.1% SDS and then were transferred to Immobilon-P transfer membranes (Millipore, Bedford, MA). Murine c-Myc, c-Jun, Bcl2, Bax, or BclXL protein was detected with murine specific anti-c-Myc (Upstate Biotechnology, Lake Placid, NY), anti-c-Jun (Santa Cruz Biotechnology, Santa Cruz, CA), anti-Bcl2 (PharMingen, San Diego, CA), anti-Bax (PharMingen), or anti-BclXL/S (Santa Cruz Biotechnology), respectively. Blots were developed in ECL reagent (Amersham, Arlington Heights, IL) according to the manufacturer’s recommended procedure.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate whether CpG DNA, a potent B cell mitogen, can also
protect resting murine spleen B cells from spontaneous apoptosis
in vitro, freshly isolated spleen B cells were treated with
a 4-log concentration range of a mitogenic 15-mer CpG O-ODN, 1916, and
a less mitogenic O-ODN with the same sequence, except that the central
cytosine was replaced by 5-methylcytosine (1936). By 40 h, 1916
drove B cells into cycle and inhibited apoptosis (measured as
hypodiploid nuclei) more effectively than 1936 (Fig. 1
A).
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A) or
16 h (not shown). Furthermore, the same half-log difference in
potency between 1916 and 1936 was seen with all three readouts (Fig. 1A). The curves were all monotonic with a width
consistent with a single affinity class of receptor. A pair of
nuclease-resistant S-ODNs were also titrated (Fig. 1B). The CpG S-ODN demonstrated a similar monotonic
curve, 200-fold greater potency, and a similar dose-response
relationship for the membrane transition as for the appearance of
hypodiploid nuclei. The minimal protective effect of the non-CpG S-ODN
at the concentrations higher than 0.1 µM (Fig. 1B)
was not sequence specific, as it occurred with all other ODN tested
that have the unnatural phosphorothioate DNA backbone (data not
shown). Relationship of ODN sequence with apoptosis and cell cycle entry
In experiments testing the effects of sequence alterations in ODNs
with phosphodiester backbones, the percentage of apoptotic
(hypodiploid) cells and percentage of cells in cycle were compared
using AO to distinguish G0 from G1 (Fig. 2). At time 0, 98% of B cells were in
G0 and about 1% had hypodiploid nuclei. As in Figure 1A, B cell apoptosis protection by ODNs was invariably
positively correlated to cell cycle entry (Fig. 2). Several active ODNs
with a variety of substitutions showed essentially indistinguishable
effects, suggesting an optimal activity level. But when the central CG
was reversed to GC (1929, Fig. 2A), activity
disappeared. Methylation of the central cytosine decreased activity
into the intermediate range (1936, Fig. 2A), whereas
methylation of the cytosine at position -4 (4 bases 5' to the central
cytosine) did not (1937, Fig. 2A). None of the
substitutions at the -2 position diminished activity (1920-2, Fig. 2B). Cytosine at position -1 substantially reduced
activity (1919, Fig. 2B), whereas other substitutions
at the same position did not (1917, 1918, Fig. 2B).
At position +1 (one base 3' of the central Guanine) a Guanine
substitution accelerated apoptosis of about half the cells and failed
to advance any cells into cycle (1925, Fig. 2C).
Other substitutions at +1 did not alter activity (1923, 1924, Fig. 2C). Substitutions at position +2 were equivalent to
each other and only slightly less active than the prototype 1916
(1926-8, Fig. 2C). Figure 2D compares
various Guanine substitutions. All the ODNs containing Guanine at +1
were inactive regardless of other Guanine substitutions (1925, 1935,
1940, 1941), and neither of the others was (1918, 1928). Interestingly,
substituting Guanines at both ends while preserving the core of the
motif significantly decreased the activity (1930, Fig. 2D). Three other ODNs with substitutions at least
three bases away from the central CG remained active (1931, 1939, and
1626, Fig. 2D).
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) at 16 h were still in G0 at 40
h. CpG ODNs rescue splenic B cells from CHX-induced apoptosis
We have shown that CHX, a protein synthesis inhibitor, at 10
µg/ml accelerates the apoptosis of murine mature spleen B cells in
vitro and that most apoptosis-protective agents cannot protect B cells
in the presence of CHX (5, 7). To evaluate whether CpG DNA can overcome
the CHX effect on spleen B cell apoptosis, freshly prepared spleen B
cells were analyzed for membrane transition and hypodiploid nuclei
after 12 h of CpG S-ODN treatment in the presence or absence of
CHX. As previously reported (5, 7), approximately 30% of primary
splenic B cells underwent spontaneous apoptosis within 12 h in
medium alone (T12 in Fig. 3). In
contrast, less than 5% of spleen B cells treated with CpG ODN for
12 h underwent apoptosis. Simultaneous addition of CpG ODN with 10
µg/ml CHX partially rescued spleen B cells from CHX-accelerated
apoptosis, approximating the result with medium alone (Fig. 3). In
contrast, control non-CpG S-ODN (1745) did not show any significant
effect on spleen B cell apoptosis in the presence or absence of CHX,
and results were essentially the same whether the membrane transition
or hypodiploid nuclei was measured (data not shown).
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shows that the CpG S-ODN 1826
was unable to stimulate any cell cycle entry in the presence of either
3 or 10 µg/ml of CHX. However, ODN 1826 still partially protected B
cells from apoptosis acceleration by 3 or 10 µg/ml CHX in a
dose-dependent fashion, showing that apoptosis protection by ODN did
not require cell cycle entry (Fig. 4).
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The proto-oncogenes c-myc, bcl2,
bax, and bclXL previously have been
reported to be involved in the regulation of cell proliferation and/or
apoptosis of various cell types, including T and B lymphocytes
(reviewed in Refs. 20–23). To investigate the possible role of CpG ODN
in regulating the expression of these genes, we examined their mRNA
level in spleen B cells after exposure to CpG or control non-CpG ODN.
Figure 5 shows the kinetic profile of
mRNA expression of selected genes in spleen B cells after treatments
with CpG or non-CpG ODN, as assessed by RPA. The level of
c-myc mRNA was substantially increased within 30 min and
peaked at 1 to 2 h after CpG ODN treatment (Fig. 5). In contrast,
the levels of c-myc mRNA in unstimulated (0 min) or control
non-CpG DNA-treated spleen B cells gradually decreased, beginning 15
min after the initiation of the culture.
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A). In contrast, the levels of
egr-1 mRNA were increased within 15 min after CpG ODN
treatment, remained above the untreated control level (0 min) until
1 h, and then became undetectable by 2 h (Fig. 5A). CpG ODN induced increased expression of
c-jun mRNA within 2 h after treatment, in contrast to
the unchanged levels seen in medium alone or control non-CpG ODN (Fig. 5A). c-fos mRNA was present at 0 and 15
min in cultures regardless of the presence of DNA and disappeared
thereafter. Unlike c-myc, egr-1, and c-jun mRNA,
the levels of c-myb, myn, and c-Ki-ras
mRNA remained unchanged in the spleen B cells under all conditions
tested (Fig. 5A).
In spleen B cells treated with CpG ODN, the levels of
bclXL mRNA were increased at 1 h and peaked
at 2 h after the initiation of culture (Fig. 5B). In contrast, no changes in the levels of
bclXL mRNA were observed in the cells cultured
in the absence of DNA or in the presence of control non-CpG ODN (Fig. 5B). There were minimal increases in the levels of
bax mRNA in the spleen B cells cultured for 4 h in the
presence or absence of CpG or non-CpG ODN, and no significant change in
the barely detectable levels of bcl2,
fas ligand or fas mRNA (Fig. 5).
Effects of CpG ODN on the synthesis of apoptosis-related proteins
Since it has been reported that the levels of
bcl2 mRNA do not correlate well with the levels
of Bcl2 protein (24), Western blots were performed to
determine whether CpG ODN stimulation results in any changes in the
levels of proteins encoded by the mRNAs studied above. The
levels of c-Myc protein gradually decreased in the spleen B cells
cultured in medium alone (Fig. 6).
Addition of non-CpG ODN did not alter the level of c-Myc in spleen B
cells. In contrast, the levels of c-Myc increased within 1 h and
peaked at 6 h after CpG ODN stimulation. We could detect minimal
amounts of c-Jun and BclXL in the spleen B cells freshly
isolated or cultured in media alone or in the presence of non-CpG ODN.
However, the levels of both c-Jun and BclXL were greatly
increased after 6 h of stimulation with CpG ODN. As expected,
Bcl2 was constitutively produced in spleen B cells, and we
could not detect any significant differences in Bcl2 among
cells cultured in CpG or non-CpG ODN or medium alone (Fig. 6).
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, concentrations higher than 1 µg/ml S-ODN 1826 were able
to inhibit apoptosis in 10 µg/ml CHX from 86% at 16 h to about
50%, so a Western blot was run under these conditions (Fig. 7). After 16 h in 10 µg/ml CHX,
the c-Myc and Bax originally present had disappeared, but
Bcl2 was unchanged. As in Figures 6 and 7, the S-ODN 1826
increased c-Myc and induced BclXL and c-Jun. Bax was
induced by S-ODN but the BclXL/Bax ratio increased (seen
also with O-ODN, not shown). Thus, under conditions where substantial
apoptosis protection was present but cell cycle entry was prevented
(Fig. 4), there was partial preservation of c-Myc, but c-Jun and
BclXL were not detected (Fig. 7). Bax, BclXL,
and Bcl2 reverted to the pattern seen at T0 or at 16 h
in medium alone (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A signal generated through surface Ig can lead to proliferation or apoptosis depending on the maturation stage of the responding B cells. Green and Scott’s two signal: death/survival model (25) suggests that activation (signal 1) drives the cell into G1, where the choice between apoptosis and S phase entry depends upon additional progression signals from the Th cell (signal 2). Thus, signal 2 acts as an apoptosis inhibitor. In B cell lines with an immature phenotype (WEHI-231), though cells are already in G1, signal 1 (anti-Ig alone) still induces apoptosis, and CpG ODN block it (17), suggesting that they provide signal 2. The current paper explores how this action of CpG ODN applies to naïve resting B cells, and how the death/survival model can be modified to include naïve B cells.
As previously shown (5, 7, 8), mature resting spleen B cells in
G0 underwent spontaneous apoptosis in medium alone. They
did so without first entering G1 as detected by AO (Fig. 2)
or by oncogene up-regulation (Fig. 5A). Whenever CpG
ODN induced cell cycle entry, they also protected B cells from
apoptosis (Fig. 2), indicating that CpG ODN provide signals 2 and 1 to
naive B cells. Against the alternative interpretation that
G0 cells responding to ODN do not receive signal 2 until
they are in G1, Figure 4 shows that when G1
entry was prevented by inhibiting protein synthesis, apoptosis
inhibition by ODN clearly occurred in G0 cells. ODN
protection of B cells from the membrane transition (detected by MC540
flow cytometry) becomes detectable between 1 and 2 h (our
unpublished observations), well before G1 entry. Other
agents that block apoptosis in naïve cells, such as IL-4 and
CD40 ligand (8), also can act as apoptosis-blocking components of
signal 2 in activated cells. IL-4 can do so without inducing
G1 entry (8). Thus, our results suggest an interesting
extension of the two signal death/survival model to include
naïve as well as activated cells: a "signal 2" agent that
blocks apoptosis in G1 cells promotes S phase entry,
whereas when the same agent blocks apoptosis in G0 cells,
it potentiates G1 entry, manifest when the cells also
receive signal 1. Rapid G1 entry, apoptosis inhibition, and
S phase entry all occur with ODN alone (Fig. 2) because ODN provide
both signals. We are currently testing this revised model with agents
that provide only one of the two signals.
The apoptosis-protective effect of CpG ODN was dose dependent (Fig. 1)
and exquisitely sequence specific (Fig. 2). To study the sequence
specificity in detail, we used a series of O-ODNs and employed AO for
the simultaneous assessment of cell cycle and apoptosis. At 40 h,
B cells with hypodiploid DNA were observed to have RNA contents
appropriate for G0, but extending into mid-G1.
Apoptotic cells may be underestimated if many cells lose DNA in S/G2/M,
if they are aggregated (aggregates are gated out), or if they lose
enough RNA to pass into the debris gate, but they may be overestimated
if excessive cellular fragmentation occurs. Pilot experiments enabled
us to set the gates for both PI and AO to minimize these sources of
error. In B cells, we developed conditions for measuring hypodiploid
nuclei with PI that agree with the percentage of DNA fragmented
(5).
Spontaneous apoptosis and failure to exit G0 were observed
with calf thymus DNA as with medium alone (Fig. 2A).
The prototype active phosphodiester ODN, 1916, profoundly inhibited
apoptosis while stimulating cell cycle entry. Generally, the
apoptosis-protective and cell cycle-progressive effects of ODN were
proportional. It was striking to observe how closely the other active
ODNs duplicated the effects seen with the prototype 1916, namely: 1)
rapid advance of about 55 to 65% of B cells into G1 by
16 h; 2) further advance of 20% of B cells into S phase between
16 and 40 h, with only 10% more advancing from G0 to
G1; and 3) only 5 to 10% entering apoptosis by 40 h,
compared with 50% in medium alone. Thus, at 40 h, 15 to 20% of B
cells exposed to active ODNs remained in G0 without
entering apoptosis (non-B cell contamination is less than
5%).
The mitogenic activity of E. coli DNA has been attributed to
its 20-fold greater content of unmethylated CpG pairs relative to
mammalian DNA (10). That E. coli DNA is less potent on a
weight basis than CpG ODN (Fig. 2A) is consistent
with the dilution of active sequences with inactive sequences in whole
DNA, though the existence of sequences that block the CpG effect
remains an interesting possibility. In vertebrate genomes, the most
common bases that precede or follow CpG dinucleotides are cytosine and
guanine, respectively (26). It is therefore noteworthy that these are
exactly the same flanking bases that abolish the stimulatory activity
of CpG dinucleotides (Fig. 2, B and C),
suggesting the possibility that their presence in vertebrate DNA may
function to prevent autoactivation by self-DNA.
Judging from enzyme and Ag prototypes, a hypothetical ODN-receptor
protein would be expected to bind an ODN at multiple points. Single
amino acid substitutions that disrupt protein binding usually involve
side chains, which either possess radically different size or
chemistry, or which cause a major local conformational change. But with
ODNs, although Figure 2 provides several examples of single base
changes that failed to alter ODN activity, there are also examples in
which changes as minor as substitution of one pyrimidine for another
(1917 vs 1919 in Fig. 2B), or one purine for another
(1923 vs 1925 in Fig. 2C) caused a change from full
activity to inactivity. Reversal of the central CG to GC had the same
effect (Fig. 2A). Addition of one methyl group to the
central cytosine decreased activity by half (Fig. 2A). These extreme positive and negative changes in
activity with minimal structural change provide stringent criteria to
be met by the ODN-recognizing entity. In the simplest case, one might
anticipate that the ODN-recognizing entity might have binding affinity
for ODNs that would correspond to the functional data in this paper,
and so these data have guided our ongoing effort to identify and
characterize this entity.
Although sequence is clearly important, receptor binding might also be
affected by ODN conformational change, implying folding of a short ODN
to form secondary structures. In the prototype ODN, 1916, bases at
positions +3 and +4 could base pair with -5 and -6 to form a loop,
and alternatively +5 and +6 could pair with -3 and -4. However, ODN
1939 is modified to disrupt one potential folding mode, and ODNs 1931
and 1626 are modified so as to disrupt both of these potential folding
modes, yet retain full activity (Fig. 2D). ODN 1930
with polyguanine ends retains about half of its activity, despite the
lack of palindromic pairs (Fig. 2D). Some of the
substitutions that prevent the single base pairing of the adenine at
-1 with thymidine at +1 fail to disrupt activity (Fig. 2, B
and C). These results do not support ODN conformation
as a significant variable in determining activity. In the limited
comparisons made to date (not shown), changing from phosphodiester (the
natural form, as in Fig. 2) to nuclease-resistant phosphorothioate
(Figs. 1, 3–7) altered the potency but not the specificity.
Surprisingly, the dose-response curves for the effects of CpG ODN on
both apoptosis protection and cell cycle entry (Fig. 1) were steep and
uniform enough to be compatible with the hypothesis that both
downstream effects are determined entirely by interaction of ODN with a
uniform population of DNA-binding sites obeying the mass action law,
without additional variables. In contrast, the sequence nonspecific
apoptosis inhibition curve of non-CpG S-ODN (example in Fig. 1B) was much broader.
DNA cleavage and the membrane transition detected by MC540 staining are
events that differ in their biochemistry, timing, and intracellular
localization, yet both were substantially and equally prevented by
active ODN (Figs. 1B and 3). Thus, in B cells undergoing
spontaneous apoptosis, the inhibitory influence of ODN is proximal to
both the membrane transition (loss of plasma membrane phospholipid
asymmetry) and DNA cleavage.
Several genes have been reported to be associated with activation or
inhibition of cell proliferation or apoptosis. CpG DNA rescues WEHI-231
B cells from anti-IgM-induced growth arrest and apoptosis and
up-regulates c-myc and bclXL
expression (17). The product of proto-oncogene c-myc is an
important regulator of proliferation and is sufficient to cause the
G0-G1 transition (reviewed in 27 .
However, continued expression of c-Myc in the presence of cell cycle
inhibitors or in the absence of apoptosis inhibitors leads to death by
apoptosis (28, 29). Therefore, c-Myc favors either proliferation or
apoptosis, depending on the cell type and status of other growth
signals. The levels of both c-myc mRNA and protein were
decreased in spleen B cells when exposed to medium alone or non-CpG
ODN, while they were up-regulated by CpG ODN (Figs. 5 and 6). Increased
c-Myc may lead B cells to exit from G0 into G1
phase (30, 31) and eventually to S-phase entry (32, 33) through
cooperation with other molecules involved in cell cycle progression
and/or survival, such as Egr-1, c-Jun, and Bcl2 family
members. Indeed, CpG ODNs strongly induced expression of
c-jun mRNA within 2 h and induced transient increases
in the level of egr-1 mRNA (Fig. 5A), as
well as increased c-Jun protein by 6 h (Fig. 6). These changes are
associated with cell growth in response to a variety of stimuli
(34, 35, 36). In particular, Jun protein is a component of the
transcription factor AP-1, which regulates the transcription of a
number of genes, including other growth factors and cytokines (reviewed
in 37 .
Bcl2 has been reported to oppose c-Myc-driven apoptosis
without affecting the c-Myc mitogenic function (38, 39, 40). Enforced
expression of a bcl2 transgene prevents or
delays apoptosis of B cells (8, 41). Both bcl2
mRNA and protein were constitutively expressed in spleen mature B cells
(42, Figs. 5 and 6) and neither were affected by CpG DNA (
Figs. 5–7).
This constitutively expressed Bcl2 is not sufficient for
providing a survival signal for mature B cells because after 16 h
in 10 µg/ml CHX, 85% of B cells are apoptotic (Fig. 4), despite
unchanged levels of Bcl2 (Fig. 7). However, CpG ODN also
produced a major increase in bclXL mRNA (Fig. 5B) and protein (Figs. 6 and 7), which were not
detectable in fresh spleen B cells. Overexpression of
bclX protects WEHI-231 B cells from
anti-IgM-mediated apoptosis and prolongs the survival of peripheral
B cells in vivo and in vitro (43, 44). Furthermore, when costimulation
by CpG DNA or CD40 ligation rescues WEHI-231 cells from
anti-IgM-induced apoptosis, bclXL expression
is increased (14, 15, 17, 45). BclXL, but not
bcl2, is also induced in peripheral B cells in
the presence of anti-IgM, anti-CD40, or LPS (44, 46). When
cultured in medium alone, normal mature B cells do not express
detectable bclXL mRNA or protein (Ref. 44;
Figs. 5–7). CpG ODN, but not control non-CpG ODN, rapidly induced increases
in bclXL mRNA and protein (
Figs. 5–7). Even
though bax mRNA and protein were increased by CpG ODN, the ratio of
BclXL to Bax increased (Figs. 5B and 7),
favoring survival (47). These results suggested the possibility that
CpG ODN prevented apoptosis and induced proliferation by inducing
concomitant expression of c-Myc and BclXL.
Mechanisms by which CpG ODNs induce B cell proliferation, secretion of
Ig and several cytokines, and protection of WEHI-231 cells from
BCR-mediated growth arrest and apoptosis are still under investigation.
Our previous studies indicated that B cells do not have a specific
membrane receptor for CpG ODN. ODN uptake by cells, while required for
activation, is not different between active and inactive ODN (10). In
addition, CpG ODN itself does not induce any change in protein
phosphorylation, inositol trisphosphate generation, or Ca2+
flux within 10 min after treatment in splenic B cells, or CH12.LX, a
murine B cell line (A.M.K., unpublished observation). The earliest
event we have been able to detect in primary B cells and B cell lines
after CpG DNA treatment was an increase in the generation of
intracellular reactive oxygen species (ROS), which leads to the
transcription of several genes such as c-myc, IL-6, IL-12,
and TNF-
via activation of
NF
B4 (13, 48). These
intracellular ROS contribute to the CpG ODN-mediated protection against
anti-IgM-induced growth arrest and apoptosis of WEHI-231 cells
(48), and we are investigating whether this may also be true in mature
B cells. Such a relationship would contrast with the observation that
ROS favor apoptosis and antioxidants inhibit apoptosis in other
cells (49, 50).
The transcriptional activation of c-myc by NFB may be an
especially important step. CD40 ligand, which resembles ODN in
providing both survival and proliferation signals to normal B cells,
also shares the ability of ODN (48) to activate c-myc
transcription through NFB in WEHI-231 cells (51, 52).
Anti-µ-induced apoptosis in WEHI-231 cells is accompanied by a drop
in c-Myc (51), whereas Myc stabilization prevents apoptosis (52).
Figure 5, A and B, show that when resting B cells
were cultured in medium alone (leading to spontaneous apoptosis; 5 , c-myc mRNA declined sharply between 1 and 2 h,
whereas exposure to CpG (but not non-CpG) DNA increased
c-myc mRNA levels.
Further evidence supporting a role for c-Myc preservation in apoptosis
protection by ODN derives from our studies with the protein synthesis
inhibitor CHX. Several apoptosis-protective agents (IL-4, IFN-, PMA)
are unable to protect B cells from apoptosis in the presence of the
protein synthesis inhibitor CHX (Peckham, Stunz, and Ashman,
unpublished observations), but ODNs were partially protective (Figs. 3 and 4), even if CHX was added 1 h before ODN (data not shown). We
were able to find conditions where no cell cycle entry occurred, yet
partial apoptosis protection was evident (Fig. 4). Under these
conditions, c-Myc was conspicuously preserved, although neither
BclXL nor c-Jun induction was detected (Fig. 7). These
results are consistent with the view that c-Myc preservation is
sufficient for at least partial apoptosis protection in B cells (52).
They also suggest that CpG DNA may somehow inhibit the normally rapid
degradation of c-Myc protein.
The observation that CpG ODN can partially protect B cells from
apoptosis acceleration by CHX under conditions where it cannot restore
cell cycle entry shows that cell cycle entry is not required for
apoptosis protection by ODN (Fig. 5). PMA, IL-4 (5), 0.1 µg/ml of CHX
(8), and elevated levels of Bcl2 (41) are also able to
protect B cells from apoptosis without causing cell cycle entry.
Figures 4 and 7 also provide an example of rapid induction of apoptosis
(by CHX) despite there being no change in the levels of the long-lived
Bcl2.
In summary, our results demonstrate that CpG motifs, which are common
in bacterial DNA, rescue mature spleen B cells from spontaneous
apoptosis and induce spleen B cell cycle entry in a sequence-specific
manner. CpG ODNs provide both signals 1 and 2, probably by directly
and/or indirectly up-regulating genes involved in cell cycle
progression and survival such as c-myc and
bclXL. The exquisite sequence specificity (Fig. 2) and the evidence that downstream events are controlled by a
homogenous population of ODN-recognizing sites (Fig. 1) have obvious
applications to our ongoing effort to identify and characterize the
mechanism of ODN recognition within the cell.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Robert. F. Ashman, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242. E-mail address:
3 Abbreviations used in this paper: ODN, oligodeoxynucleotide; O-ODN, phosphodiester oligodeoxynucleotide; S-ODN, phosphorothioate oligodeoxynucleotide; CHX, cycloheximide; PI, propidium iodide; MC, merocyanine; AO, acridine orange; RPA, RNase protection assay; ROS, reactive oxygen species.
4 A.-K. Yi, R. Tuetken, T. Redford, M. Waldschmidt, J. Kirsch, and A.M. Krieg. 1998. CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species. J. Immunol. In press.
Received for publication May 6, 1997. Accepted for publication February 19, 1998.
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