and IL-6 production but not degranulation from murine bone marrow-derived mast cells
Departments of Microbiology & Immunology and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
Correspondence: Dr. J. S. Marshall, Department of Microbiology and Immunology, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7 Canada. E-mail: Jean.Marshall{at}Dal.ca
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(TNF-) by mast cells treated with the CpG-containing
oligodeoxynucleotides. The cytokine levels induced were directly
related to the number of CpG within a given length of sequence.
Treatment with oligonucleotides containing 3CpG induced an eightfold
increase in TNF production over control incubated mast cells. Other
cytokines, including granulocyte-macrophage colony-stimulating factor,
IL-4, interferon-
, and IL-12 were not induced by oligonucleotide
treatment. Neither CpG containing oligodeoxynucleotides nor control
oligodeoxynucleotides induced degranulation of mast cells. Bacterial
DNA from Escherichia coli also induced IL-6 from mBMMC but
neither calf thymus DNA nor methylase-treated E. coli DNA
had such an effect. Examination of the uptake of Texas red-labeled CpG
and non-CpG-containing oligodeoxynucleotides revealed that they were
both similarly taken up by the mBMMC. These results have important
implications for the mechanism by which mast cells respond to bacteria
and for the potential role of mast cells in DNA vaccination.
Key Words: DNA • cytokines • inflammation • bacteria
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(TNF-), interferon (IFN)-, -ß, and - [2
3
4
,
6
7
8
9
10
11
]. However, mammalian DNA and synthetic DNA
containing no unmethylated CpG motifs are not able to stimulate these
immune effector cells [1
, 4
,
5
]. These observations have lead to the development of
CpG-containing oligonucelotide sequences for use as adjuvants
[12
]. Mast cells have a unique role in both innate and acquired immunity [13 ]. These cells are widely distributed throughout the body, particularly at mucosal sites and the skin, at the front line of host defense. The strategic location of mast cells makes them ideal sentinel cells to initiate host response mechanisms [14 , 15 ]. Animal models of bacterial infection, employing mast cell-deficient W/WV and control mice, have provided convincing evidence that mast cells play a crucial role in host defense against bacterial infection [16 , 17 ]. Certain bacteria have been shown to be able to directly stimulate mast cells to release histamine [18 , 19 ], whereas complement-mediated mechanisms account for further mast cell activation in response to bacteria [20 ]. Previous work from this laboratory has demonstrated that bacterial products, such as lipopolysaccharides (LPS) and cholera toxin, can induce cytokine production by rat mast cells [21 , 22 ]. However, the effects of bacterial DNA and CpG-containing oligodeoxynucleotides on mast cells have not previously been reported.
In this study, we have examined the hypothesis that mast cells can
recognize and respond to bacterial DNA and CpG-ODN by releasing
inflammatory mediators. The stimulatory effects of bacterial DNA and
CpG-ODN on murine bone marrow-derived mast cells (mBMMC) have been
determined by examining degranulation through measurement of the
release of ß-hexosaminidase and through evaluation of the secretion
of cytokines. Cytokine studies have focused primarily on IL-6 and
TNF-, which are known to be associated with inflammation and can be
produced by activated rodent mast cells with or without concurrent
degranulation [20
21
22
23
24
]. To gain further insight into the
mechanisms by which CpG-ODN exert their effects, we examined
oligodeoxynucleotide uptake by mBMMC through the use of flow cytometry
and confocal microscopy.
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Table 1. Sequences of Synthetic Oligodeoxynucleotides Used in this Study
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mBMMC or MC/9 cells at 1 x 106 cells/mL were exposed
to bacterial DNA or synthetic oligodeoxynucleotides at the designated
concentrations or medium (RPMI 1640 supplemented with 10%
heat-inactivated FCS, 100 U/mL penicillin, 100 µg/mL streptomycin) as
a diluent control for 20 min or for times up to 24 h at 37°C.
Calcium ionophore, A23187 (Sigma) was used as at a range of
concentrations (see Results for individual experiments) as a positive
control. The supernatants were harvested at different time points, and
IL-6 and TNF- levels were measured by B9 bioassay and L929 bioassay,
respectively. Some of the samples were further examined for the levels
of IL-4, IL-12, IFN-, and GM-CSF by enzyme-linked immunosorbent
assay (ELISA).
B-9 bioassay for IL-6
IL-6 bioactivity was measured by B-9 hybridoma proliferation
assay [26
]. Briefly, B-9 cells were cultured in RPMI
1640 medium supplemented with 5% FCS, 100 U/mL penicillin and 100
µg/mL streptomycin, 50 µM 2-ME, and a supernatant source of IL-6.
The IL-6 assay was performed in triplicate for each sample and
standards in microtiter plates (Nunclon Inter-Med, Nunc, Roskilde,
Denmark). After a 72-h culture of B-9 cells (2500/well) with samples
and standards, 10 µL/well of 0.5% MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium;
Sigma] was added, followed by 50 µL/well of 10% Triton/HCl. The
plates were read at 550 nm on an ELISA reader. IL-6 values were
expressed as units per milliliter where one unit is equivalent to
approximately 0.45 pg/mL IL-6. The limit of detection for IL-6 was 10
U/mL. The specificity of the IL-6 assay was confirmed by use of a
neutralizing anti-murine IL-6 antibody (a gift from Dr. J. Gauldie,
Hamilton, Ontario) This antibody consistently blocked over 90% of the
induced IL-6 signal in supernatants from 3CPG-ODN-activated mBMMC (data
not shown). Bacterial DNA and synthetic oligodeoxynucleotides were
checked for their effects on B-9 cell proliferation in the presence and
absence of IL-6 standards. These nucleic acid products, at the
concentrations used in this study, had no significant effects on the
B-9 bioassay.
L929 cytotoxicity assay for TNF- bioactivity
TNF- was measured by a cytotoxicity bioassay with the
use of a TNF--sensitive, mouse fibroblast cell line, L929 (ATCC no.
CRL-2148). The method is a modification of a widely used method
[27
], and has been described in our previous studies
[21
]. Briefly, 50 µL/well of 5 x 105
L929 cells/mL in RPMI 1640 medium, supplemented with 5% FCS, 100 U/mL
penicillin, and 100 µg/mL streptomycin, were added to a 96-well
flat-bottom plate (Costar, Corning, NY), and incubated at 37°C for
18 h. The medium was discarded by suction and replaced with 50
µL/well of fresh medium containing 20 µg/mL of cycloheximide
(Sigma) and 100 µg/mL of soybean trypsin inhibitor (Sigma).
Recombinant mouse TNF- (PharMingen, San Diego, CA) was used as a
standard with seven 10-fold serial dilutions from 20,000 pg/mL in the
same medium. Fifty microliters per well of either standards or samples
were added in duplicate, and the plates were incubated at 37°C. After
an 18-h incubation, 10 µL/well of MTT (5 mg/mL) was added for a
further 4-h period of incubation. Then, 50 µL/well of
phosphate-buffered saline (PBS) pH 7.4, containing 50% N,
N-dimethylformamide (Caledon Laboratories, Edmonton, Canada)
and 20% sodium dodecyl sulfate (SDS; Bio-Rad, Mississauga, Canada) was
added, and the plate was read 550 nm after overnight incubation at
37°C. The concentrations of TNF- in the samples were calculated
with SoftMaxPro (Molecular Devices) based on the standard curves.
Pre-incubation of selected positive samples with neutralizing
anti-mouse TNF- antibody (Genzyme) completely abrogated their
cytotoxicity, confirming the specificity of the bioassay for TNF-
present in the samples. The addition of bacterial DNA or synthetic
oligodeoxynucleotides at the concentrations used in our experiments did
not significantly alter the TNF- standard curves.
Short-term mediator release and ß-hexosaminidase assay
mBMMC cells (1 x 106/mL) in modified
HEPES-Tyrode’s buffer were incubated for either 20 min or 6 h at
37°C in the presence or absence of bacterial DNA or CpG-ODN at
designated concentrations with calf thymus DNA and non-CpG containing
ODN as negative control, and 0.5 µM A23187 as positive control, then
the cells were centrifuged at 300 g for 10 min at 4°C.
After collection of supernatant, the pellets were resuspended in the
original volume of the buffer and disrupted by sonication. The modified
HEPES-Tyrode’s buffer was prepared as follows (in mM): Na, 137;
glucose, 5.6; KCl, 2.7; NaH2PO4, 0.5;
CaCl2, 1; HEPES, 10; plus 0.1% BSA, pH 7.3.
ß-Hexosaminidase assay was carried out using a previously reported method [28 ]. Briefly, 50 µL of supernatant and pellet samples in duplicate were incubated with 50 µL of 1 mM p-nitrophenyl-N-acetyl-ß-D-glucosaminide (Sigma) dissolved in 0.1 M citrate buffer, pH 5.0 in a 96-well microtiter plate at 37°C for 1 h. The reaction was stopped with 200 µL/well of 0.1 M carbonate buffer, pH 10.5. The plate was read at 405 nm in an ELISA reader. The net percent of ß-hexosaminidase release was calculated as follows: ß-hexosaminidase in supernatant/(ß-hexosaminidase in supernatant + ß-hexosaminidase in pellet) x 100.
Cytokine ELISA and mRNA assays
Mouse IL-4, TNF-, and GM-CSF levels in the samples were
measured with commercially available ELISA kits (R & D Systems,
Minneapolis, MN), and mouse IL-12 (p70) levels were measured using
ELISA kits from Amersham Pharmacia Biotech (Little Chalfont, UK). The
minimum detectable levels of mouse IL-4, TNF-, IL-12, and GM-CSF
were 8, 12, 8, and 8 pg/mL, respectively. Mouse IFN- levels in the
experimental samples were measured using an in-house ELISA method with
paired antibodies purchased from PharMingen. The ELISAs for the
cytokine followed our previously published protocol
[20
]. Briefly, the in-house ELISA involved coating wells
of a 96-well NUNC-ImmunoTM plate (Nalge Nunc International,
Nunc, Roskilde, Denmark) with anti-mouse cytokine antibody at 2 µg/mL
for 16–20 h at 4°C. Nonspecific binding to the plates was blocked
using a 1% BSA, 0.1% Tween 20 solution in PBS for 1 h at room
temperature. Fifty microliters per well of recombinant cytokine
standards and samples were added to the plate and incubated for 18–20
h at 4°C. Biotinylated anti-mouse cytokines at 0.5 µg/mL were added
to each well and incubated 2 h at 37°C. This was followed with
50 µL/well of a 1/2000 dilution of streptavidin-alkaline phosphatase
solution (Life Technologies) 30 min at room temperature, and detection
of alkaline phosphatase signal using a commercial ELISA amplification
system (Life Technologies) according to the manufacturer’s
instructions. Using this system, the minimum detectable level for mouse
IFN- was 16 pg/mL. Semiquantitative reverse transcriptase polymerase
chain reaction (PCR) was performed on RNA isolated after 3 h
incubation with 2CpG-ODN (50 µg/mL) or control-ODN. MRNA
levels for IL-6 and TNF- were assessed as previously described
[24
] using ß-actin MRNA to normalize for RNA content.
Densitometry was performed on ethidium bromide-strained agase cells.
CpG-ODN binding and internalization assays
For CpG-ODN binding assays, 50 µL of mBMMC at a concentration
of 1 x 107 cells/mL were incubated at 4°C with 2
µM of Texas red-labeled 3CpG-ODN and 3GpC-ODN in RPMI 1640/10%
heat-inactivated FCS with or without 40 µM of unlabeled 3CpG-ODN or
20 mM of EDTA. Texas red-labeled 3CpG-ODN and 3GpC-ODN were synthesized
by Life Technologies, with the same sequences as described for
unlabeled 3CpG-ODN and 3GpC-ODN. To minimize internalization of the
oligodeoxynucleotides, the incubation was carried out in the medium
containing 15 mM NaN3. After a 30-min incubation, the mast
cells were washed with cold PBS/2% BSA three times by centrifugation
at 1000 g at 4°C for 8 min. Cells were fixed in 1%
paraformaldehyde/PBS/0.1% NaN3, and examined for cell
surface-bound oligodeoxynucleotides by flow cytometry.
For CpG-ODN internalization assays, 50 µL of mBMMC at a concentration of 1 x 107 cells/mL were incubated at 37°C with 0.2–2 µM of Texas red-labeled 3CpG-ODN and 3GpC-ODN in RPMI 1640/10% FCS with or without 200 µM unlabeled 3CpG-ODN. After a 6-h incubation, the un-internalized ligands were stripped by incubating the mast cells in 0.2 M acetic acid (pH 2.5) on ice for 10 min, and washed in cold PBS/2% BSA three times by centrifugation at 1000 rpm at 4°C for 8 min. Cells were fixed in 1% paraformaldehyde/PBS/0.1% NaN3, examined by flow cytometry and confocal microscopy.
Flow cytometry and confocal microscopy
The uptake of Texas red-labeled oligodeoxynucleotides by mBMMC
after either 20-min binding assay or 6-h internalization assay were
examined on a FACScan flow cytometer (Becton Dickinson Immunocytometry
Systems, Mountain View, CA). The acquisition was done with 10,000
events per sample. The list mode data were corrected for
autofluorescence and analyzed by using Winlist 3.0 software packages
(Verity Software House, Topsham, ME).
Statistical analysis
The response of samples of the same initial preparations to
different treatments was compared using a Student’s t test
for ß-hexosaminidase, IL-6, TNF-, and other cytokines.
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and IL-6 production from mBMMC
 View larger version (36K): [in a new window] |
Figure 1. IL-6 production by mBMMC in response to stimulation with
oligodeoxynucleotides containing a single CpG motif. mBMMC were
incubated at 37°C in the presence of 0.1–100 µg/mL of synthetic
oligodeoxynucleotides with (CpG-ODN) or without 1CpG motif
(Control-ODN). As a negative control, the cells were incubated with
medium alone (Medium). Supernatants were collected at 24 h and
assessed for IL-6 by B9 bioassay. 1CpG-ODN induced significantly higher
levels of IL-6 production compared with medium control at doses of 10
mL (P < 0.05) and 100 µg/mL (P <
0.01), whereas Control-ODN did not. Bars represent mean data ±
SE. n = 4. *P < 0.05, **P < 0.01 when
compared with Medium group. Results are representative of at least
three similar experiments.
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 View larger version (24K): [in a new window] |
Figure 2. Time course of IL-6 production by mBMMC treated with 50 µg/mL of
oligodeoxynucleotides with (CpG-ODN) or without (control ODN) a single
CpG motif. As control, the cells were incubated in parallel with medium
alone (Medium). Supernatant samples were taken at different intervals
of time, and assessed for IL-6 levels using the B-9 bioassay.
Significantly higher levels of IL-6 were produced by the cells treated
with CpG-ODN than with Control-ODN or medium alone at 24 h time
point, but not at earlier time intervals. Bars represent mean data ± SE. n = 4.
***P < 0.001 when compared
with Medium group. ###P < 0.001 when
compared with ODN group. Results are representative of at least four
similar experiments.
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levels. As was the case for IL-6, there
was a dose-dependent increase in TNF- production by mBMMC in
response to 1CpG-ODN but not to control-ODN (Fig. 3
). 1CpG-ODN significantly increased TNF- production by mBMMC at
doses of 1, 10, and 100 µg/mL (P < 0.05,
P < 0.01, and P < 0.05,
respectively), whereas control-ODN at 100 µg/mL did not induce
increased TNF- production (P > 0.05). The time
course of TNF- production by mBMMC treated with 1CpG-ODN was
slightly different from that of IL-6 under the same treatment
(Fig. 4
). The peak levels of TNF- bioactivity in supernatants reached
at the 6-h time point compared with the 24-h time point, at which IL-6
reached peak levels. In view of the potential for trimeric, bioactive
TNF- to dissociate within the cultures we also examined the time
course of TNF production through the use of an ELISA method. These
studies revealed very similar kinetics of TNF- production to those
observed using the more sensitive bioassay technique (data not shown).
At the 6-h peak of response, by ELISA determination, mBMMC treated with
50 mg of 1CPG-ODN produced a mean of 29 ± 6.2 pg/mL TNF-
(n = 4), whereas levels in control-treated cells were
undetectable by this technique.
 View larger version (24K): [in a new window] |
Figure 3. TNF- production by mBMMC in response to stimulation of CpG-ODN.
mBMMC were incubated at 37°C in the presence of 0.1 to 100 µg/mL of
synthetic oligodeoxynucleotides with (CpG-ODN) or without 1CpG motif
(Control-ODN). As a negative control, the cells were incubated with
medium alone (Medium). Supernatants were collected at 24 h and
assessed for TNF- levels by L929 cytotoxicity assay. CpG-ODN induced
significantly higher levels of TNF- production at the doses of 1
µg/mL (P < 0.05), 10 µg/mL (P <
0.01), and 100 µg/mL (P < 0.05), whereas Control-ODN
at 100 µg/mL did not induce production of this cytokine
(P > 0.05). Bars represent mean data ±
SE. n = 4. *P < 0.05, **P < 0.01 when
compared with Medium group. Results are representative of at least four
similar experiments.
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 View larger version (36K): [in a new window] |
Figure 4. Time course of TNF- production by mBMMC treated with 50 µg/mL of
oligodeoxynucleotides with (CpG-ODN) or without CpG motifs
(Control-ODN). As controls, the cells were incubated in parallel with
medium alone (Medium). Supernatant samples were taken at different
intervals of time, and assessed for TNF- bioactivity by L929
cytotoxicity assay. Significantly higher levels of TNF- were
detected in supernatants of the cells treated with CpG-ODN than with
Control-ODN or medium alone at the 6-h time point, but not at other
time intervals. Bars represent mean data ± SE.
n = 4. *P < 0.05,
**P < 0.01 when compared with
Control-ODN group. Results are representative of three similar
experiments.
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produced is related to the number of
CpG sequences within the oligodeoxynucleotide sequence measurement. As shown in Figure 5A
, all three CpG-containing oligodeoxynucleotides substantially
increased IL-6 levels from mBMMC, whereas control-ODN at the same dose
had no effects on the cytokine production. The IL-6 production from
cells treated with 1CpG-ODN, 2CpG-ODN, and 3CpG-ODN were significantly
higher than that from cells treated with medium alone
(P < 0.01, 1CpG; P < 0.001, 2CpG and
3CpG respectively). However, oligodeoxynucleotides containing a greater
number of CpG dinucleotide sequences appeared to be more potent in
inducing IL-6 production from mBMMC. IL-6 production from cells treated
with 2CpG-ODN was slightly higher than that treated with 1CpG-ODN,
although this difference was not statistically significant. IL-6
production from cells treated with 3CpG-ODN was significantly higher
than that from mBMMC treated with 1CpG-ODN (P < 0.01)
or with 2CpG-ODN (P < 0.01).
 View larger version (36K): [in a new window] |
Figure 5. The amount of IL-6 (A) and TNF- (B) production induced by synthetic
oligodeoxynucleotides are related to the numbers of CpG motifs present
in the sequences of the oligodeoxynucleotides. mBMMC were treated for
24 h with 50 µg/mL oligodeoxynucleotides of with one, two, or
three CpG motifs or without CpG motifs (No CpG-ODN). The CpG-containing
oligodeoxynucleotides have the same length and similar sequences but
different numbers of CpG motifs. (A) Oligodeoxynucleotides containing
one, two, and three CpG motifs (1CpG-ODN, 2CpG-ODN, and 3CpG-ODN,
respectively) all induced significantly higher levels of IL-6
production from mBMMC than that from cells treated with medium alone
(Medium; P < 0.01, P < 0.001,
P < 0.001, respectively), whereas non-CpG-containing
oligodeoxynucleotides did not. IL-6 production from 3CpG-ODN-treated
cells was significantly higher than from 1CpG-ODN-, and
2CpG-ODN-treated cells (P < 0.01, and
P < 0.01, respectively). (B) Oligodeoxynucleotides
containing one, two, and three CpG motifs (1CpG, 2CpG, and 3CpG,
respectively) all induced significantly higher levels of TNF-
production from mBMMC than from cells treated with medium alone
(Medium; P < 0.01, P < 0.001,
P < 0.001, respectively), whereas non-CpG-containing
oligodeoxynucleotides did not. TNF- production from 3CpG-ODN-treated
cells was significantly higher than from 1CpG-ODN-, and
2CpG-ODN-treated cells (P < 0.01, and
P < 0.05, respectively).
**P < 0.01,
***P < 0.001 when compared
with CpG group. Results are representative of at least three similar
experiments.
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by
mBMMC treated with similar ODN with or without 1, 2, or 3 CpG. All the
CpG-containing ODN induced significantly higher TNF- levels from
mBMMC than was produced by cells treated with medium alone
(P < 0.01 for 1CpG-ODN, P < 0.001 for
2CpG-ODN, and P < 0.001 for 3CpG-ODN). The control-ODN
(3GpC containing sequence) at the same dose did not have a significant
effect on TNF- production by mBMMC (Fig. 5B)
. Furthermore, TNF-
production from cells treated with 3CpG-ODN was significantly higher
than that from cells treated with either 2CpG-ODN or 1CpG-ODN
(P < 0.05, and P < 0.01,
respectively). These data demonstrate that the number of CpG
dinucleotides in the oligodeoxynucleotide sequence is a contributing
factor in determining stimulatory effects of the DNA products on mast
cells.
An examination of the mRNA context of cells treated with 3CpG-ODN or
control-ODN for 3 h was performed. These experiments revealed a
mean 2.7-fold increase in TNF- mRNA (n = 3, data not
shown) but no consistent elevation in IL-6 mRNA levels
(n = 3). The latter finding was consistent with
previous reports of a high degree of posttranscriptional regulation of
IL-6 production in mast cells [29
].
In our previous studies, a substantial amount of GM-CSF was produced by
mBMMC activated through IgE receptor cross-linking [20
],
and significant levels of IFN- were induced from mBMMC treated with
recombinant mouse IL-12 [Gupta and Marshall, unpublished results].
However, in this study, neither CpG-ODN nor control ODN induced
substantial IFN-, IL-12 (p40), or GM-CSF production (Table 2
). IL-4 is an important Th2-type cytokine, and is induced in mBMMC
activated through an IgE-dependent pathway [30
].
However, no significant amount of IL-4 was induced from CpG-ODN-treated
mBMMC in this study.
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Table 2. Examination of IL-4, IL-12, GM-CSF, and IFN- Production by CpG-ODN-Activated mBMMC
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compared with
control-ODN values of 13.5 ± 6, n = 4/group. The
IL-6 response of MC/9 cells to CpG ODN activation was more striking
than that of the mBMMC with a mean of IL-6 response of 3690 ± 420
U/mL in cells activated with 50 ng/mL 3CPG-ODN for 24 h compared
with only 210 ± 37 U/mL in supernatants from parallel
control-ODN-treated cells (see Table 3
). |
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Table 3. Cytokine Production by MC/9 Cells Activated with CpG-ODN
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and IL-6 production from mBMMC
from mBMMC, calf thymus DNA did not (see Table 4 ). Methylation of bacterial DNA or CpG-containing
oligodeoxynucleotides has been shown to abolish the stimulatory effects
of the DNA on murine B cell and macrophages, and was employed as a
further control for the possibility that DNA contaminants such as LPS
might be responsible for the observed effects on cytokine expression
[3
, 25
]. In a separate series of
experiments (n = 4), we treated mBMMC with 10 µg/mL
methylase-treated or untreated E. coli DNA for 24 h,
and found that methylation of E. coli DNA completely
abolished its stimulatory effects on IL-6 production by mBMMC. The IL-6
production from cells treated with methylase-treated E. coli
DNA was 1280 ± 120 U/mL, similar to that from cells incubated in
parallel with medium alone (1387 ± 48), but significantly lower
than that from cells incubated in parallel with untreated E.
coli DNA (2054 ± 246 U/mL; P < 0.05). |
View this table: [in a new window] |
Table 4. Cytokine Response of mBMMC to Activation with E. coli DNA
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Figure 6. The effect of CpG-ODN and ODN on mBMMC short-term release of
ß-hexosaminidase. mBMMC were incubated at 37°C for 20 min in the
presence of 100 µg/mL of synthetic oligodeoxynucleotides with one or
three CpG sequences (1CpG and 3CpG, respectively), or without CpG (No
CpG). As a positive control, the cells were incubated in parallel with
0.5 µM of calcium ionophore, A23187 (A23187), and as a negative
control, the cells were incubated with medium alone (Medium).
Supernatant and pellets were collected separately, and assessed for
ß-hexosaminidase levels. Neither CpG-containing oligodeoxynucleotides
nor non-CpG-containing oligodeoxynucleotides induced ß-hexosaminidase
release at 20 min, whereas A23187 induced significantly higher levels
of ß-hexosaminidase release. Each point represents the mean data ± SE. n = 4.
***P < 0.001 when compared
with Medium group. Results are representative of at least three similar
experiments.
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mBMMC were examined by flow cytometry after incubation with Texas red-labeled 3CpG-ODN and control-ODN for 30 min at 4°C (binding assay) or for 6 h at 37°C (internalization assay). In a binding assay, both Texas red-labeled 3CpG-ODN and control-ODN had the same percent of positively labeled mBMMC (57%) and the same mean fluorescence index [8 ], suggesting that there was no quantitative difference between 3CpG-ODN and the control-ODN in their binding to the mast cell surface.
Internalization assays were performed, which employed a similar approach to that described by Häcker et al. [31 ], no significant difference was observed between the amounts of 3CpG-ODN and control-ODN entering into mast cells. As shown in Figure 7 , 3CpG-ODN uptake was very similar to that of control-ODN. More detailed studies revealed a dose-dependent increase in internalization of Texas red-labeled 3CpG-ODN (TR-3CpG) by mBMMC (Table 5 ). mBMMC incubated with 2 µM of TR-3CpG showed a positive labeling of 84% and an arbitrary mean fluorescence index (MFI) of 27, in contrast to the positive labeling of 11% and MFI of 8 for the cells incubated with 0.2 µM of TR-3CpG. Such dose-dependent internalization was also observed in mBMMC incubated with Texas red-labeled control-ODN (TR-3GpC). mBMMC had 8 and 71% of positive labeling and 7 and 23% of MFI when incubated with 0.2 and 2 µM of TR-3GpC, respectively. Furthermore, there was no substantial difference between Texas red-labeled 3CpG-ODN and 3GpC-ODN in their short-term binding/uptake by mBMMC over 20 min as judged by the percent of positively labeled cells (11 vs. 8%) and the MFI (8 vs. 7%) at the dose of 0.2 µM, and the percent of positively labeled cells (84 vs. 71%) and the MFI (27 vs. 25%) at the dose of 2 µM.
 View larger version (27K): [in a new window] |
Figure 7. Typical graphs of flow cytometry results on mBMMC uptake of Texas
red-conjugated oligodeoxynucleotides. 3CpG-ODN and 3GpC-ODN. mBMMC at
10 x 107/mL were incubated for 6 h at 37°C
with 2 µM of Texas red-conjugated 3CpG-ODN (TR-CpG) or Texas
red-conjugated 3Gpc-ODN (TR-GpC) in the presence of 200 µM of
unlabeled 3CpG-ODN (TR-GpC-ODN + CpG-ODN). As a fluorescent
background control, the cells were incubated in parallel in medium
alone. mBMMC incubated with TR-CpG and TR-GpC show marked shifts of
fluorescent intensity over the fluorescent background control in a very
similar pattern. Results are representative of four similar
experiments.
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Table 5. Uptake of Oligodeoxynucleotides Conjugated with Texas Red by mBMMC
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Increasing evidence suggests that unmethylated CpG motifs, characteristically present in bacterial DNA, can also be recognized by the vertebrate immune system as an activating signal, with consequences for the immediate immune response [4 , 34 ]. Synthetic oligodeoxynucleotides (ODN) with immunostimulatory effects are frequently characterized by a CpG, which is an unmethylated cytosine followed by guanosine (CpG) dinucleotide, flanked by two 5’ purines and two 3’ pyrimidines, a structure commonly seen in bacterial DNA, but rarely present in mammalian DNA [1 , 3 ]. Similar but related sequences have also been shown to have immunostimulatory activity [3 ].
The results of this study clearly demonstrate that mast cells can
participate in the response to CpG containing DNA sequences through the
selective production of two cytokines associated with inflammation,
TNF- and IL-6. The amounts of CpG-containing DNA required to
activate mast cells are relatively high compared with some other cell
types [3
4
5
6
7
]. The residence of mast cells at sites that
interface with the external environment such as airways, skin, and
gastrointestinal tract brings these cells into direct contact with
pathogenic organisms at the site of invasion and colonization. It is
notable that relatively high levels of other bacterial-associated
products, such as LPS [21
], are also required to
activate mast cells compared with other effector cell populations.
The potential for cytokine responses from contaminating cells such as
macrophages in our mBMMC cultures contributing to our findings has been
carefully considered. For this reason only thoroughly evaluated very
pure (>98%) mast cell cultures were used for the cytokine studies. In
view of the evidence of preformed TNF and IL-6 within unactivated mast
cells [24
, 35
] examination of cytokine
induction within specific cells by immunohistochemical techniques was
not appropriate. Experiments using the J774 macrophage cell line, under
our experimental conditions, confirmed previous reports
[2
, 25
] that CpG-ODN would significantly
activate this cell type but the time course of macrophage TNF
production was somewhat delayed compared with our mast cell
preparations (data not shown). The strong IL-6 and TNF- response of
MC/9 cells to 3CpG-ODN further confirms the ability of mast cells to
selectively produce TNF and IL-6 in response to this stimulus.
The two cytokines induced by CpG-ODN treatment of mast cells are
critical to the regulation and mobilization of the immune response.
TNF- is known to enhance adhesion molecule expression on the
vascular endothelium and thus enhance inflammatory cell recruitment. In
the context of the lung, this cytokine can also induce
bronchoconstriction and airways hyperresponsiveness
[36
37
38
]. IL-6 is a critical cytokine in the initiation
of the acute phase response and also critical to antibody formation
through effects on plasma cell differentiation. It has recently been
suggested that this cytokine may play an anti-inflammatory role
in vivo [39
]. Based on the cytokine
production we have measured, the effects of CpG-ODN on mBMMC appear to
be highly selective. We have demonstrated that while CpG-ODN induced
significantly higher levels of IL-6 and TNF- production from mBMMC,
IL-4, IL-12, IFN-, and GM-CSF were not induced. Previous studies by
us, and others, have shown that activated mBMMC can produce substantial
amounts of all these cytokines [24
, 30
].
Bacterial DNA and CpG-containing oligonucleotides have been shown to
induce IL-6, IL-12, IFN-, TNF-, and IL-10 but not IL-2, IL-4, and
IL-5, production from murine spleen cells [40
41
42
]. The
current results demonstrate a highly selective cytokine response by the
mast cell, which does not fall into such a classical type 1 cytokine
profile.
The time course of CpG-ODN-induced cytokine production and the lack of
preformed mediator release strongly suggests that bacterial DNA
sequences can selectively induce mast cells to produce IL-6 and TNF-
without the necessity for degranulation. These observations are in
keeping with other reports of cytokine production that is independent
of degranulation by mast cells in response to bacterial products
[21
, 22
]. The time course of the response
of mast cells to CpG-containing DNA sequences is very similar to that
observed in mBMMC in response to LPS. The mBMMC IL-6 response to LPS is
similar to that published for rat PMC with peak values after 18–24 h
[21
]. In contrast, the peak levels of TNF production,
after LPS challenge, are observed at 6 h post-activation [McCurdy
and Marshall, unpublished results]. We can speculate that the ability
of CpG-ODN to generate an early TNF- response from mast cells may
aid in the recruitment of effector cells to sites of infection. The
later IL-6 response might be important to limit the scope of
inflammatory tissue damage or to encourage plasma cell development. An
increase in IL-6 production after CpG-ODN administration has previously
been observed in vivo [43
], within a similar
time frame. However, the kinetics of TNF- production were not
examined in this study. It should be noted that all of the mBMMC used
in this study were derived from a single strain of mice (57B16) and
that MC9 cells are derived from the (C57B616 x A/J) F1. This
mouse strain was selected in view of its propensity for type 1 cytokine
responses and the ease with which mast cells can be grown from bone
marrow precursors. It is possible that the results obtained in this
mouse strain may not be representative of all murine mast cells.
Data from flow cytometry and confocal microscopic studies have
demonstrated no significant difference between CpG-ODN and control-ODN
in terms of their uptake by mBMMC, although there is significant
difference between such sequences in terms of their induction of
cytokine production. These observations support the early prediction
that the major signaling mechanisms mediating the immunostimulatory
effects of bacterial and synthetic DNA occur after their uptake
[2
] and suggest that mast cells follow a similar pattern
of activation by DNA to that observed in other cell types. The close
relationship between the numbers of CpG within a given sequence and its
ability to selectively induce the production of TNF- and IL-6 and
the complete lack of cytokine response from sequences containing GpC
as controls suggests a highly specific and closely regulated
cytokine induction mechanism.
Uptake of oligodeoxynucleotides has been studied in a number of other cell types; however, there are conflicting data on whether the uptake of oligodeoxynucleotides is mediated by specific receptors on the cell surface [44 45 46 ]. Several groups have reported that labeled oligodeoxynucleotides were internalized into cells in a concentration- and time-dependent manner consistent with a pinocytotic mechanism, independent of cell-surface receptors [3 , 31 , 44 , 45 ]. Other groups reported that the internalization of oligodeoxynucleotides was, in part, mediated through DNA receptors or binding proteins on the cell surface [46 , 47 ]. Our study showed a dose-dependent uptake of CpG-ODN that could not be inhibited by a large excess of unlabeled ODN. These data are consistent with mast cells taking up ODN through a fluid-phase pinocytotic mechanism, as suggested in other cell systems, rather than a receptor-mediated endocytotic pathway. We did not observe marked differences in the degree of uptake of CpG-containing ODN and control ODN or in their pattern of cellular distribution by flow cytometry and confocal microscopy. The confocal miscroscopic examination of cells treated with Texas red-labeled 3CpG-ODN or control ODN revealed a pattern of discrete areas of peripheral cytoplasmic staining in the vast majority of cells, consistent with uptake by a pinocytotic/endocytotic pathway.
The recognition that mast cells can respond to CpG-containing sequences
has several important implications. In the context of allergic disease,
CpG-containing oligonucleotides have already been employed to enhance
the Th1-type cytokine responses with some demonstrated efficacy
[48
49
50
51
]. In one recent publication, long-term
prevention of allergic lung inflammation was achieved by administration
of CPG-ODN [51
]. The production of IL-6 by the mast cell
in response to CpG-ODN could contribute to an anti-inflammatory effect,
whereas a short-term TNF- response from mast cells may initiate
other regulatory mechanisms. The ability to target local, resident mast
cell populations through CpG activation may allow more localized
therapeutic strategies to be developed in mast cell-rich sites such as
the skin and airways. In the context of DNA-vaccination, the
observations of CpG-induced mast cell cytokine production may explain
some of the tissue-specific differences in responses to DNA vaccination
approaches. It is notable that mast cell-rich sites such as the skin
have been shown to be particularly good sites for DNA vaccination
protocols [52
]. Our observations suggest that the mast
cells’ response to CpG containing DNA sequences could play a role in
the mast cell response to bacterial infection along with other potent
mast cell activators such as complement components and LPS, although
the relative importance of this new method of mast cell activation
remains to be determined.
Received June 1, 2000; revised August 14, 2000; accepted August 16, 2000.
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