| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Life Science, Center for Biofunctional Molecules, Pohang University of Science and Technology, Pohang, Korea; and
Magnetic Resonance Team, Korea Basic Science Institute, Taejon, Korea
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IL-12 production from mouse splenic dendritic
cells (DCs). The optimal induction of cytokine production was achieved
by the addition of a hexameric dG (dG6) run. In contrast,
the existence of a dG6 run either at the 5' terminus of
phosphodiester CpG ODNs or at the 3' terminus of phosphorothioate CpG
ODNs diminished CpG-mediated cytokine induction, suggesting that the
effects of a dG run depend on its location and the chemical property of
the ODN backbone, respectively. In addition, we provided the evidence
that the conjugation of a dG6 run caused the structural
transformation of CpG ODNs, which facilitates their targeting into
mouse APCs such as splenic DCs, B cells, and peritoneal macrophages
with a scavenger receptor type A ligand specificity. Among primary
APCs, DCs were the most potent for CpG ODN-mediated IL-12 production.
Furthermore, we demonstrated that the conjugation of a dG6
run into the 3' terminus of phosphodiester CpG ODNs was crucial for
their ability to generate Th1 immunity in vivo. Thus, the conjugation
of a dG6 run into phosphodiester CpG ODNs would be an
alternative way to optimize their immunostimulatory potentials in vitro
and in vivo. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In this regard the tagging of consecutive dG residues, called a dG run (16), to CpG ODNs may modulate their immunostimulatory potential, since the dG run is known to bind to the scavenger receptor (SR) (17, 18), which is mainly expressed in the cells of monocytic lineage (19). Kimura et al. demonstrated that IFN production and NK activity from mouse splenocytes were increased by the addition of dG12 runs next to the CpG motif (AACGTT) (17). Also, the existence of poly(dG) at both ends of CpG ODN, which is partially phosphorothioate modified, could enhance NK cell activation in human PBMC, although this activity was significantly decreased when the ODN was entirely phosphorothioate modified (20). These results imply that the dG run may increase the uptake of CpG ODNs into target cells through SR and influence CpG-mediated cellular signaling. However, it is unclear which types of cells are direct targets of dG run-containing CpG ODNs. Moreover, it remains to be determined whether the dG run conjugation can modulate in vivo functions of CpG ODNs.
Here, we demonstrated that the conjugation of a dG6 run into the 3' terminus of phosphodiester CpG ODNs significantly augmented their immunostimulatory potentials in terms of IL-12 production from mouse primary APCs and the ability to generate Th1 immunity in mice. Of note, the effects of a dG6 run were dependent upon its location and the chemical property of the ODN backbone, and the conjugation of a dG6 run induced structural transformation of CpG ODNs, which facilitated their binding to DCs in an SR-A-dependent manner.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
All CpG ODNs with phosphodiester and phosphorothioate backbone were purchased from GenoTech (Taejon, Korea). Several ODNs are modified by tagging the fluorescein into the 3' or 5' terminus (f-ODNs). Endotoxin contamination measured by the Limulus assay (Sigma, St. Louis, MO) was negligible in all ODN preparations. Dextran sulfate, chondroitin sulfate, fucoidan, oleic acid, fibrinogen, and low density lipoprotein were purchased from Sigma, and anti-CD18 mAb was purchased from PharMingen (San Diego, CA). Specific pathogen-free conditioned 6-wk-old female C57BL/6 and BALB/c mice were purchased from Dae-Han Laboratory Animal Center (Eumsung, Korea) and Japan SLC (Shizuoka, Japan).
Isolation of primary APCs
Splenic DCs were isolated from female C57BL/6 mice as previously shown with minor modifications (21, 22). Briefly, splenocytes were adhered to a tissue culture plate for 90 min, and then nonadherent cells were discarded. Adherent cells were cultured overnight in the presence of 10 ng/ml of recombinant murine GM-CSF (R&D Systems, Minneapolis, MN). The weakly adherent cells were loaded on a Percoll density gradient, and low density cells from the interface were used as splenic DC-enriched populations. For the direct purification of splenic DCs, splenocytes were incubated with anti-CD11c microbeads and then passed through a column of midiMACS system (Miltenyi Biotec, Auburn, CA), and the remaining CD11c+ cells were used. Resting peritoneal macrophages were harvested as previously described (23) by washing the peritoneal cavity of C57BL/6 mice with the culture medium and were seeded in a tissue culture dish. After incubation for 9 h, nonadherent cells were removed by washing three times with medium, and remaining adherent cells were used in the following experiments. Splenic B cells were isolated from the nonadherent population of splenocytes. Total splenocytes were seeded into a tissue culture plate for 90 min, and anti-CD19 microbeads were coincubated with a nonadherent population of splenocytes. By passing through columns of the miniMACS system, CD19+ B cells were finally purified.
Delivery of ODNs
ODNs (1.5–3 µM) were incubated with various APCs for either
10 h (TNF-) or 30 h (IL-12) with RPMI complete medium
(Life Technologies/BRL, Gaithersburg, MD), which were supplemented with
10% FBS, 2-ME (50 µM), and L-glutamine (1.5 mM). Also,
ODNs (0.3 µM) were codelivered with 3 µg of cationic lipid complex,
Lipogen (BodYtech, Chuncheon, Korea), to DCs and were incubated for
4 h in serum-free RPMI medium. After washing the liposome-ODN
complexes, cells were further incubated for 24 h with RPMI
complete medium.
Cytokine detection
Various APCs were stimulated by ODNs or LPS, and the culture
supernatants were quantitated by commercial ELISA kits (Genzyme,
Cambridge, MA) for detection of both IL-12 and TNF- or by specific
mAb pairs for detection of both IFN-
and IL-4. For sandwich ELISAs,
purified anti-mouse IL-4/IFN- mAbs (PharMingen) were adsorbed to
capture cytokines on polystyrene base microtiter immunoassay plates
(Dynex Technologies, Chantilly, VA). The culture supernatants diluted
appropriately were incubated following biotinylated anti-mouse
IL-4/IFN- mAbs. Streptavidin-peroxidase (Southern Biotechnology
Associates, Birmingham, AL) was used for detection followed by
H2O2 and
tetramethylbenzidine substrate (Kirkegaard & Perry Laboratories,
Gaithersburg, MD) solutions. Recombinant IL-4/IFN- (PharMingen) were
used as standards for calculating cytokine concentrations in the
culture supernatants tested.
Binding and internalization assay with fluoresceinated ODNs
CD11c+ DCs (105), CD19+ B cells, and peritoneal macrophages were preincubated at 4°C for 15 min to suppress endocytosis and then incubated with various f-ODNs (5 µM) for 30 min at 4°C. After complete washing the cells with PBS, the mean fluorescent intensity (MFI) was measured from f-ODNs bound to the cells by FACScan (Becton Dickinson, Mountain View, CA). For detecting the internalization of ODNs, f-ODNs (1 or 5 µM) were treated for 30 min at 4°C and then further incubated for 1.5 or 6 h at 37°C. After treatment of phosphorothioate-modified poly(dC)20 to deprive cell-bound f-ODNs, the MFI from internalized f-ODNs was measured by FACScan. The MFI was measured from the live cells, extracting the dead population of cells with propidium iodide staining.
Nuclear magnetic resonance (NMR) analysis for ODN structure
ODNs were chemically synthesized on an Applied Biosystems 392 (Foster City, CA) and were purified by PAGE and size exclusion chromatography. The purified samples were dialyzed against PBS buffer. After lyophilization, the samples were dissolved in 400 µl of PBS. NMR experiments were performed on a Bruker DMX 600 spectrometer (Billerica, MA). Jump-and-return pulse was used to suppress water peak. The delay between the two 90°C pulses was set to optimize the imino proton peaks. The spectral width was 12,000 Hz (20 ppm), and 256 scans were averaged for each sample. The recycle delay was 1.8 s. The FIDs were apodized with 1 Hz exponential functions. The polynomial baseline correction was applied to the imino proton regions.
Immunizations
Six- to 8-wk-old female BALB/c mice were immunized with various Ags with or without ODNs. Briefly, 5 µg of heat-inactivated HIV type 1 (HIV-1) particles that had been formulated with aluminum hydroxide (Sigma) were s.c. immunized together with 50 µg of phosphodiester ODNs. We also used gDE2t protein (24), which is a fusion protein of herpes simplex virus type 1 glycoprotein D (gD) and C-terminal truncated hepatitis C virus (HCV) envelope protein 2 (E2t) for the immunization. Two micrograms of phosphodiester (1826D, M21) or phosphorothioate (1826T) ODNs were i.m. administered with 5 µg of gDE2t protein. Four weeks after the first immunization, mice were given booster immunizations with same immunization regimens. Humoral and cellular immune responses were monitored 2 wk after booster immunization. HIV- or HCV E2t-specific IgG responses were determined by ELISA. For the detection of cytokine expression from CD4+ T cells from immunized mice, CD4+ T cells were finally purified from nonadherent population of splenocytes using anti-CD4 microbeads and the miniMACS system. CD4+ T cells (2 x 105) were stimulated with E2t proteins purified from Chinese hamster ovary (CHO) cells for 5 days in the presence of 105 syngeneic APCs. APCs were isolated from the adherent population of splenocytes from naive BALB/c mice and then treated with mitomycin C (Sigma).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To investigate the effects of a dG run on the cytokine induction
by phosphodiester CpG ODNs, we sought the bacterial DNA sequence
containing a dG run as well as a CpG hexamer nucleotide motif. The
20-mer ODN derived from ampR gene within the pUC19 sequences
(nucleotides 2293–2312) (25), designated p19, was tested
for its ability to induce IL-12 and TNF- from splenic DCs (Table I
). The putative dG run
(GGGGCG)-containing phosphodiester CpG ODN p19 elicited higher levels
of cytokine production, comparable to those induced by LPS. However,
deletion of the putative dG run from p19 (p19d) completely abrogated
cytokine production. As expected, the induction of cytokine production
by p19 was entirely dependent upon the presence of a CpG sequence (M1).
In addition, changes in nucleotides within the putative dG run from dG
to dC (M7, M13, M14, and M15) resulted in dramatic reduction of IL-12
and TNF- production, but substitution of a dC with a dG (M16) caused
a marked increase in the production of cytokines, implying that the dG
run is critical for cytokine induction by p19. However, the
introduction into the 5' terminus of a dG run of various lengths (M19,
M26, and M27) did not enhance cytokine induction. To explore the
optimal length of a 3'-dG run, we tagged various numbers of dG residues
to the 3' terminus of p19d (Fig. 1). It
is likely that the 3'-dG run should have at least four consecutive dG
residues for cytokine induction. The maximum induction of IL-12 and
TNF- production was achieved by the addition of a
dG6 run (p19d-dG6; M16). In
addition, the ability to induce cytokines was retained, but slightly
decreased, when the longer dG runs (dG8,
dG10, and dG12) were added.
The conversion of CpG dinucleotide to GpC
(p19d-dG12/GC) completely abrogated cytokine
production, and poly(dG)20 did not induce
cytokine production, indicating that the dG run alone could not induce
cytokine production in the absence of a CpG motif.
|
|
To further address the effect of a dG6 run
on different sequence contexts of phosphodiester CpG ODNs, we added a
dG6 run to the 5' or the 3' terminus of two
different CpG ODNs, such as 1826D and M12, respectively (Table II). There was a significant increase in
the activity of cytokine production when a dG6
run was fused to the 3' terminus (1826D vs M21, and M12 vs M22), but
not to the 5' terminus (1826D vs M20, and M12 vs M23), suggesting that
the effect of a dG6 run depends on its location
within the phosphodiester CpG ODNs. To further determine the
specificity of a dG run, we replaced a dG6 run
with other nucleotide runs (dA6,
dT6, and dC6) and tested
for cytokine induction (Table II). M16A, M16T, and M16C were completely
deprived of the ability to induce cytokines, indicating the specificity
of a dG run for this function. To assess the effect of the dG run on
other mouse APCs, CpG ODNs conjugated with a dG6
run were treated with either peritoneal macrophages (pMac) or splenic B
cells. Although pMac and B cells produced much lower amount of IL-12
than did splenic DCs, the addition of a dG6 run
to the 3' of CpG ODNs (M21 and M16), but not into the 5' (M20 and M26),
also enhanced IL-12 production (Fig. 2).
As expected, the replacement of a dG6 run with a
dC6 run totally abolished the induction of IL-12.
These results suggest that the dG run is effective in a broad range of
cells in a position-dependent manner.
|
|
and IL-12 production compared with1826T (Fig. 3), suggesting that the conjugation of a
dG6 run at the 3' terminus inhibits
immunostimulatory potentials of phosphorothioate-modified CpG ODNs. In
particular, M21T produced lesser amounts of IL-12 but higher amounts of
TNF- than did M21, a dG6 run-containing
phosphodiester CpG ODN. These results imply that the dG run differently
affects CpG-mediated cytokine induction depending upon the chemical
property of backbone structure of CpG ODNs.
|
To determine the effect of a dG run conjugation on the cellular
binding and the internalization of CpG ODNs in
CD11c+ splenic DCs, we performed FACS analysis
using fluoresceinated phosphodiester CpG ODNs. Fluoresceinated p19
(f-p19), but not f-p19d, was shown to efficiently bind to DCs (Fig. 4A). f-M16 and f-M26 that
contain a 3' and a 5'-dG6 run, respectively,
bound more efficiently than did f-p19. The cellular binding of ODNs was
also enhanced by the tagging of a dG6 run to the
3' and 5' termini of f-1826D (f-M21 and f-M20, respectively).
Interestingly, CpG ODNs with a dG6 run at their
5' termini bound to DCs less efficiently than those with a
dG6 run at their 3' termini. However, the
substitution of a dG6 run on f-M21 with a
dC6 run (f-M21C) completely abolished its binding
activity, suggesting that the cellular binding of ODN is dG run
specific. Moreover, the capacity for internalization of ODNs was
correlated with the binding activity (Fig. 4B), implying
that the binding of the dG6 run resulted in an
increase in the intracellular concentration of CpG ODNs. It is worth
noting that the apparent ODN uptake shown in Fig. 4B may be
less than the actual uptake due to the rapid degradation of
phosphodiester ODN (26). Similar results were observed in
B cells and pMac (data not shown). In addition, pMac had approximately
4 times higher binding affinity for ODNs than B cells and DCs.
Nevertheless, the level of IL-12 production from pMac and B cells by
CpG ODNs conjugated with a dG6 run was much lower
than that from DCs (Fig. 2
and Table II). Therefore, these data suggest
that among primary APCs, DCs are the most potent cells for CpG
ODN-mediated IL-12 induction.
|
), and it was shown
that all ODNs, except M16GC, greatly increased IL-12 production.
Interestingly, CpG ODNs conjugated with a dA6, a
dT6, or a dC6 run at their
3' termini (M16A, M16T, and M16C) also induced high levels of IL-12
production comparable to M16, suggesting that the
3'-dG6 run is only necessary for the binding and
the subsequent internalization of ODNs. In contrast, there was a
significant reduction in IL-12 production by the 5' addition of a
dG6 run to CpG ODN (M20 and M23) compared with
1826D and M12 that lack a dG6 run, indicating
that the dG6 run at the 5' terminus may inhibit
the cytokine induction mediated by CpG ODNs, even if it could increase
their cellular binding and internalization activities. The dG run-containing CpG ODNs bind to the receptor with a SR-A ligand specificity on splenic DCs
It was previously suggested that ODNs containing a
dG12 run at their 5' and 3' termini could bind to
SR on macrophages (17). In fact, the expression of the SR
family in subsets of DCs has not been clearly identified. Singh et al.
showed that maleyl-BSA which is the ligand of many SR families (SR-A
I/II, SR-BI, CD36) could bind to murine splenic DCs (21).
In addition, human DCs could express CD36 (27) and Cla-1
(28), the human homologue of the murine SR-BI. Thus, we
performed the competitive binding assays using f-M21 for SR families in
DCs. The binding of f-M21 to DCs was significantly impaired by
treatment with the competitive ligands for SR-A, such as dextran
sulfate, fucoidan, and poly(dG)20
(19) (Fig. 5). In
contrast, chondroitin sulfate and poly(dC)20 did
not compete with f-M21 for binding to DCs, indicating the specificity
of competition. In addition, pretreatment with low density lipoprotein
and oleic acid, which are ligands for SR-BI and CD36, respectively, did
not diminish the binding of f-M21 even at a high concentration (0.5
mg/ml), suggesting that the receptor with a SR-A ligand specificity may
confer the binding activity to dG run-containing ODNs. It was
previously reported that phosphodiester or phosphorothioate ODNs could
bind to Mac-1 proteins (CD11b/CD18) in human polymorphonuclear
leukocytes and that fibrinogen and anti-CD18 mAb efficiently
compete with the binding of ODNs to Mac-1 (29). However,
pretreatment of fibrinogen and anti-CD18 mAb could not decrease the
binding of f-M21 to DCs and to pMac, suggesting that Mac-1 was not a
target receptor for the binding of dG6
run-containing phosphodiester ODNs, at least in the murine
APCs.
|
It was previously reported that short oligo-dGs could be assembled
into a four-stranded helix, called a tetraplex, that was stabilized by
G quartets and inhibited SR activity (18). To investigate
whether specific structural determinants of CpG ODNs are required for
binding to SR, we determined the tetraplex formation of various CpG
ODNs with or without a dG run by the presence of unusual imino proton
NMR resonances in 10–12 ppm (30, 31). The population of
tetraplex structures relative to that of Watson-Crick base-paired
hairpin or duplex structures was estimated for various ODNs (Fig. 6). All ODNs with four or longer
consecutive dG residues formed tetraplex structures regardless of their
location. Longer dG runs increased the tetraplex population. ODNs
containing a 3'-dG6 run (M16 and M21) formed more
clear tetraplex structures than did those containing a
5'-dG6 run (M16' and M20), which may explain why
CpG ODNs with a 3'-dG6 run were more efficient in
the cellular binding. For the 3'-dG run-containing CpG ODNs, the
ability to induce IL-12 appeared to correlate with the population of
the tetraplex structure, suggesting that the structural transformation
into a tetraplex was critical for cytokine induction by CpG ODNs with a
3'-dG run.
|
To determine whether the conjugation of a dG run also affected the
immunostimulatory potentials of phosphodiester CpG ODNs in vivo,
alum-absorbed, heat-inactivated HIV-1 particles with or without ODNs
were s.c. immunized into mice. Without the coadministration of ODNs,
mice raised moderate levels of IgG, which were mainly composed of IgG1
isotype (Fig. 7A). The
coadministration of a dG6 run-containing CpG ODN,
M16, increased total IgG production (p <
0.05), especially the IgG2a isotype (p <
0.03), which is one of the representatives of Th1 immune responses
(32). In contrast, the coadministration of either M16C or
M16GC did not significantly increase the levels of total IgG and IgG2a.
These results suggest that the presence of both a CpG motif and a dG
run are critical for the adjuvanticity of phosphodiester CpG ODN,
whereby either a dG run or a CpG motif alone did not confer an
immunostimulatory effect in vivo. Since only phosphorothioate-modified
CpG ODNs have been used as adjuvants to date (33, 34, 35), we
next compared the adjuvanticity of dG6 run-tagged
phosphodiester CpG ODN (M21) with phosphorothioate CpG ODN (1826T) for
the generation of Th1 immunity. We also used the phosphodiester CpG ODN
(1826D) as a control CpG ODN for these experiments. To clearly observe
the adjuvanticity, we immunized gDE2t fusion proteins (24)
in the absence of alum together with relatively low dose of ODNs (2
µg) into mice. The coimmunization of gDE2t proteins with 1826D
elicited a low level of HCV E2-specific IgG2a production (Fig. 7B). In contrast, coadministration of M21 elicited IgG2a
production approximately 50-fold higher than did 1826D, but 2-fold
lower than that induced by 1826T, indicating that the tagging of a
dG6 run into 1826D enhances its adjuvanticity to
elicit IgG2a production. In concordance with IgG subtype responses,
CD4+ T cells from mice that received either M21
or 1826T had a higher ratio of IFN- to IL-4 secretion than did mice
that received 1826D by the stimulation with recombinant HCV E2t
proteins (Fig. 7C). Overall, our results suggest that the
conjugation of a dG run is critical for induction of Th1 immunity to
protein Ags when phosphodiester CpG ODNs are used as adjuvants.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene on murine macrophages
(36). In contrast, it is known that SR ligation by
maley-BSA selectively suppresses LPS-induced IL-12 production in bone
marrow-derived macrophages (37). However, it is unlikely
that SR ligation through G quartets by itself affects intracellular
signaling for cytokine induction in DCs, since neither TNF- nor
IL-12 is induced by either dG6 run-containing
ODNs that lacked a CpG motif or poly(dG)20.
Formation of a tetraplex structure stabilized by G quartets
dramatically affected both the efficiency of binding and subsequent
internalization of dG run-containing CpG ODNs into APCs. It is unclear
why a dG6 run conjugated at the 5' terminus is
less efficient at inducing a tetraplex structure than a
dG6 run conjugated at the 3' terminus.
Nevertheless, the location of a dG6 run within
CpG ODNs appeared to be important for their functions, since a
5'-dG6 run inhibited cytokine induction elicited
by CpG motifs when CpG ODNs were delivered with liposomes. Our results
are partially consistent with a recent report by Krieg et al.
(38) that demonstrated that some ODNs containing
self-complementary and/or G-rich sequences (CpG-N) neutralized the
ability of CpG motif-mediated cytokine induction in cis or
trans. They also suggested that the higher ordered structure
of CpG-N might exert neutralizing effects on cytokine induction.
However, our structural data showed that a G quartet structure failed
to induce IL-12 production only when it was located at the 5' terminus
of phosphodiester CpG ODNs (Fig. 6). The detailed mechanisms by which a
5'-dG6 run inhibits CpG-mediated cytokine
induction should be explored.
Previous reports suggested that the sequence contexts flanking the CpG
motif were important for the activity of CpG ODNs (10, 38, 39), but it was unclear how and why the flanking sequences
influenced their functions. Even though 1826D induced a relatively low
level of cytokine production from DCs, other phosphodiester CpG ODNs
tested in this study had no activity in the absence of a dG run.
However, these CpG ODNs elicited cytokine production similar to that by
1826D when they were delivered with liposomes (Table II), suggesting
that the specificity of ODN sequences may be related to their
internalization efficiency. In addition, it is likely that the length
of the CpG ODN affects the efficiency of intracellular signaling for
cytokine induction and/or the stability of the ODN, since 11-mer CpG
ODN (M12) induced IL-12 much less efficiently than did longer CpG ODNs
when delivered with liposomes (Table II). Taken together, our results
suggest that both the sequence context and the length of phosphodiester
CpG ODNs are important to modulate their immunostimulatory potentials.
The chemical property of the backbone structure of CpG ODNs also
affected their ability to induce cytokine. Of note, the conjugation of
a dG6 run at the 3' terminus of a
phosphorothioate CpG ODN decreased its ability for both TNF- and
IL-12 induction from splenic DCs. Ballas et al. also demonstrated that
the poly(dG) ends completely abrogated the enhancement of NK activity
by CpG motifs when the entire backbone was phosphorothioate modified
(20). Although additional experiments are required to
clarify whether the backbone structure influences CpG-mediated cytokine
induction, the phosphorothioate modification is suggested to reduce the
binding affinity of the CpG ODN to a putative cellular target
protein(s) (40).
The intrinsic immunostimulatory potential of the phosphorothioate backbone can generate Janus-faced consequences, since it strongly boosts cytokine induction elicited by CpG motifs and induces detrimental immune activation and toxicity (1, 41, 42, 43). In fact, we observed that a single s.c. administration of phosphorothioate, but not phosphodiester, CpG ODNs (30–50 µg) with 5 µg of gDE2t protein absorbed in alum elicited granuloma formation at the injection sites in mice (data not shown). In contrast to phosphorothioate CpG ODNs, phosphodiester CpG ODNs without a dG run were poorly immunostimulatory by themselves in vitro and in vivo, possibly due to the low efficiency of nonspecific endocytosis and vulnerability to nucleases. However, the conjugation of a dG run may surmount the defects of phosphodiester CpG ODNs due to the following reasons. First, since the tetraplex structure was reported to confer serum nuclease resistance (44), it is possible that the conjugation of a dG run may extend a half-life of phosphodiester CpG ODNs. Second, the dG run efficiently targets CpG ODNs into various APCs via the SR. Our data demonstrated that the conjugation of a dG6 run into phosphodiester CpG ODNs can generate Th1 immunity in vivo. In addition, we observed that the binding and internalization of phosphodiester ODNs into HepG2 cells that express SRs were significantly increased in the presence of a dG run (unpublished observations), suggesting that the conjugation of a dG run has a great potential for delivery of ODNs into cells expressing SRs, such as hepatocytes (45, 46). In conclusion, it is likely that the conjugation of a dG6 run into phosphodiester CpG ODNs would be a promising modification for therapeutic purposes of CpG ODNs.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 S.W.L. and M.K.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Young Chul Sung, Department of Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea.
4 Abbreviations used in this paper: ODN, oligodeoxynucleotide; DC, dendritic cell; SR, scavenger receptor; f-ODN, ODN modified by tagging the fluorescein into the 3' or 5' terminus; MFI, mean fluorescent intensity; NMR, nuclear magnetic resonance; gD, glycoprotein D; HCV, hepatitis C virus; pMac, peritoneal macrophages.
Received for publication April 26, 2000. Accepted for publication July 13, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-dependent mechanisms. Proc. Natl. Acad. Sci. USA 96:6970.. Proc. Natl. Acad. Sci. USA 93:2879.vß5 and CD36, and cross-present antigens to cytotoxic T lymphocytes. J. Exp. Med. 188:1359. and B cell stimulatory factor-1 reciprocally regulate Ig isotype production. Science 236:944.
B binding, and transcription of the TNF- gene in murine macrophages. J. Leukocyte Biol. 60:784.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  S. Ivanov, A.-M. Dragoi, X. Wang, C. Dallacosta, J. Louten, G. Musco, G. Sitia, G. S. Yap, Y. Wan, C. A. Biron, et al. A novel role for HMGB1 in TLR9-mediated inflammatory responses to CpG-DNA Blood, September 15, 2007; 110(6): 1970 - 1981. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Jozefowski, T. H. Sulahian, M. Arredouani, and L. Kobzik Role of scavenger receptor MARCO in macrophage responses to CpG oligodeoxynucleotides J. Leukoc. Biol., October 1, 2006; 80(4): 870 - 879. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Osawa, S. Iho, R. Takauji, H. Takatsuka, S. Yamamoto, T. Takahashi, S. Horiguchi, Y. Urasaki, T. Matsuki, and S. Fujieda Collaborative Action of NF-{kappa}B and p38 MAPK Is Involved in CpG DNA-Induced IFN-{alpha} and Chemokine Production in Human Plasmacytoid Dendritic Cells J. Immunol., October 1, 2006; 177(7): 4841 - 4852. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gursel, I. Gursel, H. S. Mostowski, and D. M. Klinman CXCL16 Influences the Nature and Specificity of CpG-Induced Immune Activation J. Immunol., August 1, 2006; 177(3): 1575 - 1580. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kerkmann, L. T. Costa, C. Richter, S. Rothenfusser, J. Battiany, V. Hornung, J. Johnson, S. Englert, T. Ketterer, W. Heckl, et al. Spontaneous Formation of Nucleic Acid-based Nanoparticles Is Responsible for High Interferon-{alpha} Induction by CpG-A in Plasmacytoid Dendritic Cells J. Biol. Chem., March 4, 2005; 280(9): 8086 - 8093. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. N. Wu, J. Lee, E. Raz, M. Corr, and D. A. Carson Necessity of Oligonucleotide Aggregation for Toll-like Receptor 9 Activation J. Biol. Chem., August 6, 2004; 279(32): 33071 - 33078. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Lundberg, P. Welander, X. Han, and E. Cantin Herpes Simplex Virus Type 1 DNA Is Immunostimulatory In Vitro and In Vivo J. Virol., October 15, 2003; 77(20): 11158 - 11169. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. D. Marshall, E. M. Hessel, J. Gregorio, C. Abbate, P. Yee, M. Chu, G. V. Nest, R. L. Coffman, and K. L. Fearon Novel chimeric immunomodulatory compounds containing short CpG oligodeoxyribonucleotides have differential activities in human cells Nucleic Acids Res., September 1, 2003; 31(17): 5122 - 5133. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Marshall, K. Fearon, C. Abbate, S. Subramanian, P. Yee, J. Gregorio, R. L. Coffman, and G. Van Nest Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions J. Leukoc. Biol., June 1, 2003; 73(6): 781 - 792. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Hemmi, T. Kaisho, K. Takeda, and S. Akira The Roles of Toll-Like Receptor 9, MyD88, and DNA-Dependent Protein Kinase Catalytic Subunit in the Effects of Two Distinct CpG DNAs on Dendritic Cell Subsets J. Immunol., March 15, 2003; 170(6): 3059 - 3064. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gursel, D. Verthelyi, I. Gursel, K. J. Ishii, and D. M. Klinman Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide J. Leukoc. Biol., May 1, 2002; 71(5): 813 - 820. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Park, S. W. Lee, and Y. C. Sung Cutting Edge: CpG DNA Inhibits Dendritic Cell Apoptosis by Up-Regulating Cellular Inhibitor of Apoptosis Proteins Through the Phosphatidylinositide-3'-OH Kinase Pathway J. Immunol., January 1, 2002; 168(1): 5 - 8. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Mui, S. G. Raney, S. C. Semple, and M. J. Hope Immune Stimulation by a CpG-Containing Oligodeoxynucleotide Is Enhanced When Encapsulated and Delivered in Lipid Particles J. Pharmacol. Exp. Ther., September 1, 2001; 298(3): 1185 - 1192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. H. Baek, S. J. Ha, and Y. C. Sung A Novel Function of Phosphorothioate Oligodeoxynucleotides as Chemoattractants for Primary Macrophages J. Immunol., September 1, 2001; 167(5): 2847 - 2854. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
