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Institute for Molecular Bioscience and Departments of Microbiology and Biochemistry, University of Queensland, Brisbane, Australia
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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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Phosphodiester oligodeoxynucleotides (PO-ODN) containing CpG motifs mimic the direct effects of native bacterial DNA in vitro, including activation of macrophages, dendritic cells, and B lymphocytes. The short t1/2 of PO-ODN in vivo (11) precludes their use in therapy. It is relevant to ask whether the more stable PS-ODN mimic all of the actions of native phosphodiester CpG DNA. Recognition of the differences, and their molecular basis, may direct the identification of more effective mimetics of bacterial DNA for clinical application. There have been few comparative studies of CpG PS- and PO-ODN. Published observations vary depending on the cell system used and the effector function measured. A CpG PS-ODN was effective at promoting survival of human dendritic cell precursors from peripheral blood at lower concentrations than a CpG PO-ODN (12), but the length and sequence of the respective PS-ODN and PO-ODN were not identical. Studies by Boggs et al. (13) using sequence identical PS-ODN and PO-ODN demonstrated that for NK cell activation PO-ODN were less active than corresponding PS-ODN, but the situation was reversed when a transfection reagent, lipofectin, was added. Several studies indicate that PS-ODN are more potent than corresponding PO-ODN at inducing B cell proliferation (14, 15, 16, 17). Studies on B cell proliferation are complicated by the fact that human and mouse B cells can respond to PS-ODN in a CpG-independent manner (18, 19). In addition, if using thymidine uptake as a measure of proliferation, PO-ODN degradation can dilute the radioactive thymidine pool (20). PS-ODN have also been found to be more potent in induction of spleen cell IL-12 and CD69 expression (15).
CpG DNA mimics many of the actions of bacterial LPS on murine
macrophages, including activation of NF-
B-dependent transcription
and release of cytokines such as TNF-
, IL-6, and IL-12
(21, 22, 23, 24, 25). In this study, we use a range of assays of
macrophage activation to compare the actions of PS-ODN and PO-ODN of
identical sequence. The results show that the two types of ODN have
distinct activities and PS-ODN do not function merely as a more stable
version of active PO-ODN.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The murine macrophage-like cell line, RAW264, was obtained from the American Type Culture Collection (Manassas, VA). Bone marrow-derived macrophages (BMM) were obtained by cultivation of femoral bone marrow cells for 1 wk on bacteriological petri dishes in recombinant human macrophage CSF (CSF-1) (a gift from Chiron, Emeryville, CA), as described previously (26). The medium in all experiments was RPMI 1640 plus 10% heat-inactivated FBS with 20 U/ml penicillin and 20 µg/ml streptomycin. Serum was screened for low LPS contamination using RAW264a4 cells. RAW264a4 is a stable transfectant of RAW264 with an integrated HIV-1 LTR-luciferase reporter gene that is activated by low concentrations of LPS, or CpG DNA (21, 27).
PO-ODN and PS-ODN were purchased from Geneworks (Adelaide, Australia) or Oligos Etc. (Wilsonville, OR). AO-1, a PO-ODN, has the sequence 5'-GCTCATGACGTTCCTGATGCTG-3', which is similar to the oligo 1668 of Yi et al. (28), with the addition of terminal G residues. NAO-1 is the same sequence with the core CG (underlined) reversed to GC. AOS-1 and NAOS-1 are the corresponding PS-ODN. AAC-22 (29) is slightly less active than AO-1 and has the sequence ACCGATAACGTTGCCGGTGACG. SAAC-22 is the phosphorothioate version of AAC-22. Results obtained with AO-1 and AAC-22 or their corresponding phosphorothioate versions were similar and they could be used interchangeably in this work. For uptake experiments, AO-1 and AOS-1 were purchased with a Cy3 fluorophore attached at the 5' end. LPS from Salmonella minnesota Re595 was purchased from Sigma (St. Louis, MO) and was dissolved as a 10 mg/ml stock solution in 0.1% triethylamine and sonicated. Fluorescein-labeled anionic dextran, 10,000 m.w., was purchased from Molecular Probes (Eugene, OR).
Luciferase assays
For the HIV-1 LTR assay, 2.5 x 105 RAW264a4 cells were placed in 1 ml of medium in a 24-well plate overnight. After reduction of the total volume to 0.5 ml, cells were incubated with stimuli for 2 h, unless stated otherwise. Cells were harvested and assayed for luciferase activity, as described (21, 27), except that the assays were conducted using a Trilux plate luminometer and reagents provided by Packard Instruments (Meriden, CT). For analysis of IL-12 promoter activity, the murine IL-12 p40 promoter, as described by Cowdery et al. (25), was amplified by PCR and subcloned into the pGL2 luciferase vector by Dr. E. Gold (University of Washington School of Medicine, Seattle, WA) RAW264 cells were transfected with 10 µg of pGL2-IL12p and 2 µg of the selectable plasmid pNeo/Tak (30), as described (31), and subjected to selection in 200 µg/ml G418 for 2 wk. Approximately 200 colonies appeared on the dishes. These were harvested and pooled, and grown for a further week in G418 before assay. Inducible luciferase activity has been maintained thereafter for several months in culture. For the IL-12 promoter assay, 105 cells were incubated in 1 ml medium overnight, followed by addition of ODN for 6 h, then lysis and assay as above.
Nitrite assay
The assay of nitrite using the Griess reagent was used as an
indicator of NO production by both BMM and RAW264, as described
previously (23). Briefly, 4 x
104 RAW264 or 105 BMM (with
104 U/ml CSF-1) were plated in 96-well plates in
200 µl supplemented RPMI and cultured overnight. Cells were then
pretreated with 20 U/ml murine rIFN-
for 2 h (R&D Systems,
Minneapolis, MI), followed by addition of the triggering stimulus.
After 24 h, supernatants were removed and assayed for nitrite.
Cellular viability assay
The reduction of the dye MTT (Sigma) was used to measure relative number of viable cells. BMM were harvested and plated in CSF-1-free medium for 18 h (5 x 104 cells/well in 200 µl in a 96-well plate). The desired additions were made, and the cells were incubated for an additional 48 h before assessment of MTT reduction, as described previously (32).
Down-modulation of surface CSF-1R
BMM were starved of CSF-1 overnight, then incubated with the desired treatments on bacteriological petri dishes, before being immunostained for surface expression of CSF-1R using the specific mAb AFS98, as described previously (32).
Phosphorylation of ERK-1/2
Cell extracts and Western blotting for the determination of the phosphorylation state of the extracellular signal-regulated kinases 1 and 2 (ERK-1/2) were conducted as described previously (32).
Intracellular TNF- assays
For the plate assay, RAW264 cells were plated at
105 cells/well in 96-well plates and incubated
overnight. To increase cellular retention of TNF-, brefeldin A (10
µg/ml) was added 15 min before incubation with ODN for 2 h.
Cells were washed once in ice-cold PBS and fixed with 20% acetone,
0.02% BSA in PBS for 2 h at 4°C. Cells were washed three times
with PBS, blocked for 1 h with 1% BSA in PBS, and then incubated
for 1 h with 1/4000 dilution of rabbit anti-mouse TNF- Ab
(Genzyme, Cambridge, MA) in PBS/1% BSA. Cells were washed three times
with 0.2% Tween 20 in PBS, for a total of 20 min, then incubated for
1 h with 1/1000 dilution of secondary Ab (anti-rabbit HRP
conjugate; New England Biolabs, Beverly, MA) in PBS/1% BSA. Secondary
Ab was removed and cells were incubated for 30 min in 5% goat serum in
PBS/1% BSA. Cells were washed three times with 0.2% Tween 20 in PBS,
for a total of 20 min, and then once in PBS. Color development with
2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) reagent
(560 µg/ml ABTS (Sigma), 0.008%
H2O2, 50 mM citric acid,
0.1 M Na2HPO4, pH 4.2) was
measured at 415 nm.
For FACS analysis, RAW264 cells plated in 1 ml of medium were treated
with 5 µg/ml brefeldin A together with ODN for 4 h. Cells were
harvested and stained for intracellular TNF-, as described by
Underhill et al. (30).
ODN and dextran uptake
RAW264 cells were plated overnight at 250,000/well in 1 ml of medium in 24-well plates, and reduced to 0.25 ml before addition of ODN. Cy3-labeled ODN were added for the indicated times before removal of supernatant and addition of ice-cold PBA (PBS/0.1% BSA/0.1% sodium azide). Cells were harvested, and incubated on ice for 10 min with 25 mg/ml dextran sulfate in PBA to remove surface-bound ODN. Cells were then washed twice in 5–10 ml PBA and analyzed by flow cytometry on a FACScalibur (Becton Dickinson, San Jose, CA) for internalized ODN. Control samples in which cells were incubated with ODN on ice to allow cell surface binding but not internalization showed that the procedure did effectively strip off surface-bound ODN. Results were corrected for a slight difference in the degree of fluorochrome labeling of the two ODN, AO-1 and AOS-1. For fluorescein-labeled dextran uptake, cells were processed as for ODN uptake experiments, except incubation with dextran sulfate was omitted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophages primed with IFN- respond to exposure to CpG DNA
with expression of inducible NO synthetase mRNA and increased NO
production (21, 23). PO-ODN and PS-ODN of the same
sequence (AO-1 and AOS-1) were compared for their ability to induce NO
production in IFN-primed BMM and RAW264 cells in Fig. 1
, A and B,
respectively. The dose-response curves for the primary cells and the
cell line are almost indistinguishable. Both ODN were active, but AOS-1
was much more potent with responses detectable at 
100-fold lower
concentration than for AO-1. The PS-ODN AOS-1 always gave a lower
maximal activity than AO-1. High AOS-1 concentrations caused NO
production to decline from the maximum. A second pair of PO- and
PS-ODN, AAC-22 and SAAC-22, gave a similar difference in potency, and
inhibition of response at high PS-ODN concentration (result not shown).
CpG specificity was preserved for the response to AOS-1, although low
levels of NO production were observed occasionally with the PS-ODN
NAOS-1 (see Fig. 11), which differs only in having a reversal of the
CpG motif to GpC.
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priming, we considered the possibility that IFN sensitized specifically
to PS-ODN. We have shown elsewhere that CpG DNA can prevent apoptosis
that occurs in BMM deprived of the macrophage growth and survival
factor, CSF-1 (32). Fig. 2
compares the dose-response curves for the antiapoptotic activity of
AO-1 and AOS-1. Assessment of viable cell number was based upon
reduction of the dye MTT by the mitochondrial enzyme succinate
dehydrogenase. Because this assay is sensitive to factors that alter
mitochondrial respiration, we have previously validated it for this use
by showing a good correlation between MTT reduction and direct cell
count (32). AO-1 and AOS-1 both maintained cell viability
in the absence of CSF-1, whereas the corresponding GpC transversions
lacked activity. In comparative dose-response curves, AOS-1 was
30-fold more active than AO-1 (Fig. 2). The dose-response curves for
AOS-1 and AO-1 were similar in the cell viability and NO assays,
suggesting that IFN- does not sensitize preferentially to
phosphorothioate CpG DNA. As both the NO assay and the antiapoptotic
assay involve incubation for extended periods in culture, the first
explanation considered for the higher activity of PS-ODN was their
stability relative to PO-ODN. We therefore examined several shorter
term assays, in which a smaller difference in activity of PS- and
PO-ODN might be expected. Stimulation of an IL-12 promoter-reporter
construct in RAW264 cells for 6 h gave a similar threshold for
response to PO-ODN as the previous assays (Fig. 3). Again, the PS-ODN was active at much
lower concentrations and had a lower maximal activity than the
PO-ODN.
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In previous work, we have measured NF-B-dependent transcription
in macrophages after 2-h exposure to CpG DNA, using a RAW264 cell line
with an integrated HIV-1 LTR luciferase reporter construct
(21). Using this assay (Fig. 4A), the PO-ODN AO-1 gave a
similar dose-response curve to the longer term NO and antiapoptotic
assays (Figs. 1
and 2). By contrast, the corresponding PS-ODN, AOS-1
was almost inactive. The same pattern was observed with the PO-ODN
AAC-22 and its corresponding PS-ODN SAAC-22 (Fig. 4B). Both
PS-ODN caused a minimal activation of the HIV-1 LTR at low
concentration, but had little additional impact with increasing
concentration.
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, A and B). Fig. 5A shows the
representative flow-cytometric profiles. Increasing AO-1 concentration
increased the proportion of cells with completely down-modulated CSF-1R
(falling within the M1 gate). Similar to the observations made with
activation of the HIV-1 LTR, PS-ODN were much less active than their
PO-ODN counterparts. After a 1-h incubation with ODN at a concentration
of 3 µM, AOS-1 increased the percentage of cells with receptor
down-modulation from 12% to 25%, whereas with AO-1 95% of cells
fully down-modulated their CSF-1R. The same trend was observed in a
comparison of SAAC-22 and AAC-22 (Fig. 5C).
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Maintenance of cell viability in CSF-1-starved BMMs is associated
with the ability of both LPS and CpG DNA to activate the MAP kinases,
ERK-1/2 (32). LPS causes rapid activation of the MAP/ERK
kinase-1, which in turn phosphorylates ERK-1/2. A stimulatory ODN such
as AO-1 acts more slowly than LPS, perhaps reflecting the time required
for ODN internalization (unpublished observation). Fig. 6A compares the time course of
activation of ERK-1/2 in BMM by AO-1 and AOS-1. AO-1 caused maximal
phosphorylation of ERK-1/2 by 20 min, followed by a decline to an
elevated steady state after 40 min. By contrast, AOS-1 showed no
induction of ERK-1/2 phosphorylation at 20 min, and reached a peak at
1 h that was less than the maximal phosphorylation observed with
AO-1. Both ODN treatments in Fig. 6A were 3 µM. Because
AOS-1 seems to have some self-inhibitory action at this concentration
(Figs. 1 and 2), we checked whether lower concentrations could induce
earlier phosphorylation of ERK-1/2. Fig. 6B shows that
concentrations of AOS-1 between 0.1 and 1 µM were also unable to
stimulate ERK-1/2 phosphorylation at 20 min. We found a similar delay
in ERK-1/2 phosphorylation in response to AOS-1 in RAW264 cells (result
not shown).
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Delayed ERK-1/2 phosphorylation, poor activation of the HIV-1 LTR
at 2 h, and little down-modulation of the CSF-1R at 1 h by
PS-ODN suggested a slower cellular activation by PS-ODN than PO-ODN.
Cellular activation by CpG DNA apparently requires DNA uptake
(20). Ineffective activation by PS-ODN in some short-term
assays might potentially be explained by slower uptake into the cell.
Previous studies have shown PO-ODN to be more rapidly taken up than
PS-ODN in a colorectal adenocarcinoma cell line (33), but
faster uptake of PS-ODN in renal epithelial cells (34) and
B cells (35). To check whether poor responses were due to
slower uptake of PS-ODN, we measured cellular accumulation of
Cy3-labeled PS- and PO-ODN by flow cytometry. Cy3 is not pH sensitive
between pH 5 and 7.5 and is therefore unaffected by endosomal
acidification. Fig. 7 shows that PS-ODN
actually accumulated much more rapidly than PO-ODN in RAW264 cells. The
slower cellular accumulation of PO-ODN seen in this study was not due
to its extracellular degradation, because the differential uptake was
maintained in cells freshly plated in serum-free medium (result not
shown). Given that the difference in rates of uptake was apparent after
only 5 min, it is also not likely to be due to intracellular
degradation of PO-ODN and exocytosis of the Cy3 label. Thus, slow
uptake of PS-ODN is not responsible for its poor activity in some
assays. The possibility remains that PS-ODN take longer to reach the
site of detection of CpG DNA within the cell.
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Although PS-ODN did not efficiently activate macrophages in two
other assays performed after 1 and 2 h of stimulation (Figs. 4 and 5), it did induce TNF- protein in a 2-h incubation (Fig. 8A). The response to low
concentrations of PS-ODN was less sensitive than in the longer term NO
and apoptotic assays, with first responses seen at 0.1 µM rather than
0.03 µM. Nevertheless, any delay in signaling in response to PS-ODN
is insufficient to prevent induction of TNF- at 2 h. Analysis
at the single cell level using flow cytometry showed that AOS-1 induced
lower levels of TNF- in individual cells than AO-1 (Fig. 8, B and C). In addition, although all cells
responded to 3 µM AO-1, a population of cells failed to be stimulated
by the PS-ODN AOS-1. The lower peak response, and decline in activity
at high PS-ODN concentration seen in this study (Fig. 8A)
and in other assays (Figs. 1, 2, and 3) suggested that PS-ODN have an
inhibitory action at high concentration. Given this, we investigated
whether this inhibition was an inherent property of PS-ODN and whether
non-CpG PS-ODN can block the CpG response.
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Häcker et al. (36) have presented evidence that
non-CpG PS-ODN can block the response to CpG PS-ODN. Whether the same
applies for PO-ODN has not previously been addressed. Preincubation or
cotreatment with an equimolar concentration (2 µM) of the inactive
PO-ODN NAO-1 had no significant effect on activation of HIV-1
LTR-dependent luciferase activity in RAW264 cells induced by AO-1. Both
pre- and cotreatment with an equimolar concentration of the equivalent
PS-ODN NAOS-1 substantially diminished activation by AO-1 (Fig. 9A). Even a 6-fold molar
excess of the non-CpG PO-ODN NAO-1 had no effect on the response to
AO-1 at a concentration that was near saturating in biological CpG
activity (Fig. 9B). The effect of non-CpG PS-ODN was
specific in that it reduced CpG DNA-mediated activation of the HIV-1
LTR, but not activation mediated by LPS (Fig. 9A). Because
even the CpG-containing PS-ODN were inactive in the HIV-1 LTR assay, we
examined whether they might also be inhibitory. Indeed, SAAC-22 was
also able to prevent activation by AO-1 and AAC-22 (not shown). These
findings indicate that PS-ODN have a dominant repressive effect on
the ability of CpG DNA, but not LPS to activate in this assay.
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. There
was no requirement for pretreatment; in fact, the extent of inhibition
was greatest when PS-ODN were coadministered, whereas there was
little effect where it was added only 15 min after treatment with
AO-1. The lack of inhibition following delayed addition is
consistent with the pattern of activation of ERK-1/2 (see Fig. 6),
which is already maximal after 20 min. High dose inhibition observed in
the dose-response curve for PS-ODN activation of NO production could
also reflect a compromise between stimulation by the CpG motif, and
inhibition by the backbone. This possibility was investigated by
preincubating cells with non-CpG ODN. As seen in the HIV-1 LTR assay,
nonstimulatory PS-ODN blocked the response to AO-1, whereas minimal
inhibition of the LPS response was observed (Fig. 11). Responses to the PS-ODN AOS-1 and
SAAC-22 were also inhibited by non-CpG PS-ODN (result not shown). By
contrast, there was no inhibition by the corresponding non-CpG PO-ODN
(NAO-1) (Fig. 11) even at concentrations of 10 µM (result not
shown).
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IL-6, and IL-12 production in a macrophage cell line by non-CpG PS-ODN.
They also showed by fluorescence microscopy that at a concentration of
3 µM, non-CpG PS-ODN blocked the uptake of an active PS-ODN at 1
µM. We examined the effect of pretreatment with PS- or PO-ODN on the
uptake of Cy3-labeled ODN (Fig. 12).
The PO-ODN NAO-1 at 9 µM had minimal effect on ODN uptake. Increasing
concentrations of NAOS-1 reduced the uptake of Cy3-AOS-1. However,
although 9 µM NAOS-1 reduced the uptake of the PO-ODN Cy3-AO-1 by
50%, at lower concentrations NAOS-1 had no effect or was even
stimulatory. Thus, inhibition of uptake does not account for the
inhibition of activity of AO-1 seen with NAOS-1 between 1 and 3 µM
(Figs. 9 and 11). NAOS-1 did not inhibit uptake of fluorescein-labeled
dextran (Fig. 12), indicating that PS-ODN had no effect on fluid-phase
uptake in macrophages.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The phosphorothioate modification of ODN is necessary for effective
immunomodulatory effects to be seen in vivo. The general assumption is
that this is due to the increase in stability of the ODN, as PO-ODN are
rapidly degraded in vivo (11). PS-ODN also have
significantly greater stability in vitro. Matsukura et al.
(37) found the t1/2 of
PO-ODN in medium with 15% FCS to be 17 h, while PS-ODN showed no
degradation after 7 days. Another report found PO-ODN within spleen
cells was partially degraded after 4-h incubation, although PS-ODN was
intact (35). Even after 72-h incubation, PS-ODN was 90%
intact in cells and culture medium (38). If CpG PS-ODN
behaved just like PO-ODN but with greater stability, then we would
expect that long-term assays would show the greatest disparity in
activity between the two types of ODN. There would be a fairly constant
response to PS-ODN in different assays and a declining relative potency
of PO-ODN as the time of incubation increased. This is not what we have
observed. Regardless of the length of incubation, the dose response for
PO-ODN in a range of assays was fairly similar, with activity evident
at concentrations above 0.3–1 µM (
Figs. 1–5 and 8). The effects of
the phosphorothioate modification varied widely between assays.
Responses to PS-ODN were evident at 10–100-fold lower concentrations
than PO-ODN for induction of NO, activation of the IL-12 promoter, and
prevention of cell death (
Figs. 1–3). The much greater potency of
PS-ODN in these assays, and in in vivo immunomodulation is likely to be
due not only to greater stability, but also to much more efficient
uptake of PS-ODN (Fig. 7). However, CpG PS-ODN were much less potent
than PO-ODN in down-modulation of surface expression of the CSF-1R,
almost inactive in activation of the HIV-1 LTR, and gave delayed and
reduced ERK-1/2 phosphorylation (
Figs. 4–6).
The poor responses to PS-ODN seen in some of the shorter assays were
not due to slow uptake of PS-ODN. The possibility remained that PS-ODN
is slower in getting to the site of recognition of foreign DNA, which
remains uncharacterized. Endosomal release could be a rate-limiting
step for responses to CpG DNA. The effective response to PS-ODN in
induction of TNF- protein by 2 h (Fig. 8A) suggests
that this is not the major issue. In addition, extension of the HIV-1
LTR assay to 6 h failed to give efficient activation by PS-ODN,
and treatment of CSF-1-deprived BMM for 12 h with PS-ODN resulted
in only 50% of cells down-modulating surface expression of the CSF-1R
(results not shown). Thus, there seems to be a qualitatively different
response to the PS-ODN, rather than merely a delayed response. A
remaining possibility for poor activation in some assays is that PS-ODN
give a selective inhibition or lack of activation of certain signaling
pathways. Coordinated activation of signaling pathways may be important
in some responses, and a delay in activation of pathways such as the
ERK MAP kinases (Fig. 6A) may prevent responses. In
addition, a lowered level of activation of signaling pathways, as
observed for ERK-1/2 with PS-ODN, may stimulate some responses and not
others, if the responses have different thresholds for activation. A
comparison of PS- and PO-ODN activation of other pathways such as
NF-B, JNK, and p38 will be informative. Others have reported that
CpG PS-ODN can induce NF-B nuclear translocation in RAW264 cells
(25). Thus, the failure of PS-ODN to stimulate the HIV-1
LTR (Fig. 4) suggests that PS-ODN may poorly activate some of the other
transcription factors required, such as Ets, Sp-1, or PU.1 (39, 40).
Another way in which the two types of ODN vary is the
sequence-independent inhibitory activity of PS-ODN seen at high
concentrations. Non-CpG PS-ODN at concentrations of 1 µM and above
inhibited responses to both CpG PO-ODN (Figs. 9 and 11) and PS-ODN
(result not shown). This concentration coincides with the threshold
above which CpG PS-ODN exhibit a self-inhibitory activity (Figs. 1 and 2). Inhibition of responses to PS-ODN at high concentrations has been
observed previously (17). The inhibition seen in this
study was not due to an intrinsic toxicity of high concentrations of
phosphorothioate, as LPS reponses were not greatly inhibited (Figs. 9 and 11). A previous study has suggested that non-CpG PS-ODN may inhibit
CpG responses by blocking ODN uptake (36). We found some
inhibition of uptake of labeled ODN after treatment with non-CpG
PS-ODN, which was not seen with high concentrations of non-CpG PO-ODN
(Fig. 12). The degree of inhibition of uptake was not sufficient to
account for the inhibition of CpG activity. Thus, we propose that
PS-ODN have two inhibitory activities at high concentration, one that
reduces the uptake of ODN, and another uncharacterized activity
accounting for the self-inhibition seen at higher concentrations with
CpG PS-ODN.
Non-CpG PS-ODN inhibited the uptake of labeled CpG PS-ODN in a
dose-dependent manner, but was less effective at inhibiting the uptake
of labeled PO-ODN (Fig. 12). This, together with the much poorer uptake
of PO-ODN, suggests a difference in the mechanism of uptake between the
two types of ODN. In K562 human leukemia cells, PS-ODN uptake was
primarily receptor mediated at concentrations below 1 µM, with
fluid-phase uptake becoming important at higher concentrations
(41). A number of studies have looked for DNA receptors on
the cell surface, but the mechanism of ODN uptake remains to be
established (20, 42). The integrin MAC-1 (CD18/CD11b) has
been implicated as a specific receptor for ODN in neutrophils
(43). We found no evidence to support this function in
murine macrophages, in which ODN treatment did not alter the level of
MAC-1 Ag on the cell surface (32). In addition, cells from
mice deficient in MAC-1 have normal ODN uptake (20).
In preliminary binding studies with murine macrophages, we found that
PO-ODN had much lower cell surface binding than PS-ODN (result not
show). This has also been found using spleen cells (35).
Because PO-ODN binding to spleen cells is efficiently competed for by
PS-ODN (35), it is unlikely that they are binding
completely different classes of receptor. The differences in rate of
uptake, surface binding, and inhibition of uptake are consistent with
PS-ODN binding and uptake by a high affinity receptor, and much poorer
receptor-mediated uptake of PO-ODN. With low receptor-mediated uptake,
the contribution of fluid-phase uptake of PO-ODN would become
relatively more important, explaining the poor inhibition of PO-ODN
uptake by PS-ODN (Fig. 12). The absence of an effect of PS-ODN on
uptake of labeled dextran indicates that fluid-phase uptake is not
inhibited by PS-ODN (Fig. 12). Because receptor-mediated uptake is
inherently saturable, the inhibition of labeled CpG PS-ODN uptake by
non-CpG PS-ODN is likely to be due to competition for receptor binding.
Lack of inhibition by high concentrations of PO-ODN is explained by its
lower affinity for the receptor.
In addition to the inhibitory activities we have observed for PS-ODN,
sequence-independent activation has been documented. In vivo
administration of PS-ODN caused splenomegaly and B cell proliferation,
which was enhanced by the presence of CpG motifs (18).
Sequence-independent activation of human B cells (19) and
enhancement of LPS-induced TNF- production (44) are
thought to be mediated by PS-ODN interaction with cell surface
molecules. Activation of Sp1 transcription factor has also been
observed (45). Some of these effects may be explained by
the enhanced affinity of PS-ODN for binding to a wide variety of
proteins (46, 47). For example, PS-ODN binding inhibits
isoforms of protein kinase C (48), and can interfere with
binding of growth factors to their receptors (49). Such
interactions with signaling molecules could be responsible for the lack
of activation by PS-ODN of some macrophage responses to CpG DNA, as
well as the inhibitory effects we have observed at high concentration.
Another effect of the phosphorothioate modification may be to constrain
the three-dimensional structure of the ODN and affect its interaction
with the putative CpG receptor. Whether this could have different
effects on different responses, as we have observed in this study,
remains to be established. The poor activity of PS-ODN in eliciting
some responses could be somewhat sequence dependent. Krieg et al.
(17, 20) have suggested that the sequence requirements for
activity are more stringent with PS-ODN than with PO-ODN, and another
group has shown a sequence-dependent lack of activity of PS-ODN in
induction of TNF- (50). Thus, the change in ODN
structure introduced by the phosphorothioate modification seems to have
a greater effect on the immunostimulatory ability of some sequences
than others.
In summary, we have demonstrated that the phosphorothioate backbone modifies the response to activating CpG sequences in a number of ways. The phosphorothioate modification that makes ODN resistant to nuclease attack also greatly enhanced their uptake into murine macrophages. The increased stability and uptake are likely to be responsible for the higher potency of PS-ODN in some assays. However, the PS-ODN failed to efficiently stimulate some responses. Delayed or poor activation of signaling components, such as the ERK MAP kinases, may contribute to this effect. In addition, PS-ODN have a CpG-selective inhibitory action at high concentrations that is only partially accounted for by inhibition of ODN uptake. Thus, PS-ODN do not perfectly mimic a biological response to natural CpG DNA. Whether this is an advantage or disadvantage in therapeutic applications remains to be established. Other backbone modifications may give qualitatively different responses of use in immunotherapy.
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2 Address correspondence and reprint requests to Dr. Katryn Stacey, Institute for Molecular Bioscience, University of Queensland, Brisbane 4072, Australia.
3 Abbreviations used in this paper: PS-ODN, phosphorothioate-modified ODN; BMM, bone marrow-derived macrophage; ERK, extracellular signal-regulated kinase; LTR, long terminal repeat; MAP, mitogen-activated protein; ODN, oligodeoxynucleotide; PO-ODN, phosphodiester ODN.
Received for publication October 8, 1999. Accepted for publication July 6, 2000.
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), 1
µM Cy3-labeled AOS-1 (
), or 1 µM Cy3-labeled AO-1 (
) for 40
min. They were pretreated for 30 min with the indicated concentrations
of NAO-1 or NAOS-1. ODN uptake was measured by FACS analysis. Results
are representative of three experiments and show the mean of
duplicates. Error bars show the values of the duplicates. Results for
ODN uptake are shown relative to the control Cy3-AOS sample. Dextran
results were normalized to the control sample.