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(Journal of Leukocyte Biology. 2001;69:253-262.)
© 2001 by Society for Leukocyte Biology

CpG-containing oligodeoxynucleotides induce TNF-{alpha} and IL-6 production but not degranulation from murine bone marrow-derived mast cells

Fu-Gang Zhu and Jean S. Marshall

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mast cells are sentinel cells critical to the initiation of innate immune and inflammatory responses, particularly at mucosal surfaces. To fulfill this function they can be activated by several pathogen-associated stimuli to produce cytokines with or without concurrent degranulation. We examined the ability of immunostimulatory DNA sequences including CpG motifs, which are found in increased quantities in bacterial DNA, to activate mouse bone marrow-derived mast cells (mBMMC). Mast cells were treated with a range of doses of CpG-containing oligodeoxynucleotides or control oligodeoxynucleotides without CpG within their sequence. There was a dose-dependent increase in the production of both interleukin-6 (IL-6) and tumor necrosis factor {alpha} (TNF-{alpha}) 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-{gamma}, 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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DNA has long been regarded as immunologically inert, however, studies over recent years have demonstrated that bacterial DNA can activate immune cells from mouse and other mammalian species. Initial studies demonstrated that specific DNA sequences from Mycobacterium bovis were effective at inducing the production of interferons and activating natural killer cells [1 ]; a wide range of other cellular effects have since been described [1 2 3 4 5 ]. The immunostimulatory properties of bacterial DNA are mainly contributed by specific sequences such as cytosine guanine (CpG) motifs, which are present in the unmethylated state and with high frequency in bacterial DNA, compared with mammalian DNA [1 ]. Bacterial DNA and synthetic CpG-containing oligodeoxynucleotides (CpG-ODN) have been shown to directly activate B cells, macrophages, natural killer (NK) cells, and dendritic cells to proliferate, exhibit enhanced cytotoxicity, or to release cytokines, including interleukin (IL)-1, IL-6, IL-12, tumor necrosis factor {alpha} (TNF-{alpha}), interferon (IFN)-{alpha}, -ß, and -{gamma} [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-{alpha}, 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial DNA and synthetic oligodeoxynucleotides
DNA from Escherichia coli and calf thymus were purchased from Sigma (St. Louis, MO). The bacterial DNA was fragmented to 100–600 base pairs long by sonication. Synthetic oligodeoxynucleotides were all 20 base pairs long, phosphorothioate-modified, and purified by reverse-phase high-performance liquid chromatography (HPLC) by the manufacturer (Research Genetics, Huntsville, AL).The sequences of the oligodeoxynucleotides (Table 1 ) used in this study are taken from published articles [3 , 25 ], in which the CpG-ODN were shown to be able to activate murine B lymphocytes and macrophages. The endotoxin content was less than 0.03 ng/mg for E. coli DNA and the synthetic oligodeoxynucleotides used in this study, determined by the Limulus amebocyte lysate assay (Sigma). All the bacterial DNA and synthetic oligodeoxynucleotides were dissolved in saline at a stock concentration of 2 mg/mL, heat-denatured at 92°C for 10 min, then chilled on ice for 5 min before use.


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  		Table 1. Sequences of Synthetic Oligodeoxynucleotides Used in this Study

 
Mast cell source and activation
mBMMC were cultured from male C57BL/6 mice, 6–8 weeks old (Jackson Laboratories, Bar Harbor, ME), housed in the Animal Care Facility at Dalhousie University, Halifax, Nova Scotia, Canada. All experimental procedures were approved by the Animal Research Ethics Board of Dalhousie University. The mice were killed by CO2 inhalation. Intact femurs and tibias were removed from mice. The bone marrow cells were harvested by repeated flushing of the bone shaft with endotoxin-free RPMI 1640 medium (Life Technologies, Grand Island, NY). The bone marrow cell culture was established at a concentration of 106/mL in medium consisting of RPMI 1640, 10% heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µM 2-mercaptoethanol (2-ME), and 10% WEHI-3B conditioned medium as a source of IL-3. Nonadherent cells were transferred to fresh culture medium once a week. The purity of mast cells from the mouse bone marrow culture used in cytokine induction experiments was >98%, as confirmed by Toluidine blue and Alcian blue staining of cytocentrifuge preparations as well as by flow cytometric analysis of mBMMC-fluorescent labeled with antibody against mouse c-kit, a cell surface marker for mast cells (Cedarlane Laboratories, clone ACK4). ß-Hexosaminidase studies employed bone marrow mast cell preparations of lower purity (>85%). However, none of the contaminating cells are known to release this enzyme after short-term activation. MC/9, an IL-3-dependent mouse mast cell line was obtained from ATCC and maintained routinely in the presence of exogenous IL-3.

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-{alpha} 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-{gamma}, 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-{alpha} bioactivity
TNF-{alpha} was measured by a cytotoxicity bioassay with the use of a TNF-{alpha}-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-{alpha} (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-{alpha} 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-{alpha} antibody (Genzyme) completely abrogated their cytotoxicity, confirming the specificity of the bioassay for TNF-{alpha} 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-{alpha} 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-{alpha}, 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-{alpha}, IL-12, and GM-CSF were 8, 12, 8, and 8 pg/mL, respectively. Mouse IFN-{gamma} 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-{gamma} 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-{alpha} 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-{alpha}, and other cytokines.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CpG-ODN induces TNF-{alpha} and IL-6 production from mBMMC
CpG-ODN have been shown to directly activate B cells, macrophages, and dendritic cells, resulting in cytokine production. To examine the stimulatory effects of CpG-ODN on mBMMC, cytokine secretion from these cells was measured. mBMMC at 1 x 106cells/mL were treated with ICpG-ODN (5’-TCCATGACGTTCCTGATGCT-3’) or control-ODN (5’-TCCATGAGCTTCCTGATGCT-3’) at a range of doses from 0.1 to 100 µg/mL. Supernatants were harvested after 24-h culture, and examined for IL-6 levels. There was a dose-dependent increase in IL-6 production by mBMMC in response to CpG-ODN but not to control-ODN (Fig. 1 ). IL-6 production by mBMMC treated with 10 µg/mL (1490 ± 128 U/mL, P < 0.05) and 100 µg/mL (3122 ± 670 U/mL, P < 0.01) was significantly higher than that from cells treated with medium alone (980 ± 118 U/mL). mBMMC treated with 100 µg/mL of control-ODN produced a similar amount of IL-6 (900 ± 95 U/mL) as that of medium control. The time course of IL-6 production by mBMMC treated with CpG-ODN or control-ODN was then examined. As shown in Figure 2 , there was a significant elevation of IL-6 production by CpG-ODN-treated mBMMC at the 24-h time point (P < 0.001). IL-6 production from cells treated with control-ODN was similar to that of cells treated with medium alone (P > 0.05) at all time points examined.



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  		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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  		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.

 
Supernatants from 1CpG-ODN and control ODN-treated mBMMC were also taken to measure the TNF-{alpha} levels. As was the case for IL-6, there was a dose-dependent increase in TNF-{alpha} production by mBMMC in response to 1CpG-ODN but not to control-ODN (Fig. 3 ). 1CpG-ODN significantly increased TNF-{alpha} 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-{alpha} production (P > 0.05). The time course of TNF-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} (n = 4), whereas levels in control-treated cells were undetectable by this technique.



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  		Figure 3. TNF-{alpha} 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-{alpha} levels by L929 cytotoxicity assay. CpG-ODN induced significantly higher levels of TNF-{alpha} 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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  		Figure 4. Time course of TNF-{alpha} 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-{alpha} bioactivity by L929 cytotoxicity assay. Significantly higher levels of TNF-{alpha} 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.

 
The amount of IL-6 and TNF-{alpha} produced is related to the number of CpG sequences within the oligodeoxynucleotide sequence
Published data by other investigators [3 ] indicated that the number of CpG sequences in oligodeoxynucleotides may contribute to the stimulatory effects of CpG-ODN; we sought to examine the cytokine production of mBMMC in response to CpG-ODN of same length but with different numbers of CpG in their sequences. mBMMC at 1 x 106 cells/mL were treated for 24 h with 50 µg/mL of CpG-ODN with 1, 2, and 3 CpG dinucleotides in their sequence (1CpG-ODN, 2CpG-ODN, and 3CpG-ODN, respectively) as employed by previous investigators [3 ], and supernatants were collected for IL-6 and TNF-{alpha} 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).



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  		Figure 5. The amount of IL-6 (A) and TNF-{alpha} (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-{alpha} 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-{alpha} 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.

 
A similar trend was observed in the pattern of production of TNF-{alpha} by mBMMC treated with similar ODN with or without 1, 2, or 3 CpG. All the CpG-containing ODN induced significantly higher TNF-{alpha} 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-{alpha} production by mBMMC (Fig. 5B) . Furthermore, TNF-{alpha} 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-{alpha} 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-{gamma} 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-{gamma}, 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-{alpha} Production by CpG-ODN-Activated mBMMC

 
IL-3-dependent mast cell line MC/9 responds to CpG-ODN activation
As further confirmation that mast cells rather than contaminating cells were responsible for the observed cytokine response to CpG-ODN, we performed a series of experiments using the MC/9 mouse mast cell line 3CpG-ODN at a dose of 50 ng/mL for 24 h induced the production of a mean of 130 ± 11 pg/mL of TNF-{alpha} 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

 
Bacterial DNA induces TNF-{alpha} and IL-6 production from mBMMC
The ability of CpG-containing oligodeoxynucleotide sequences to induce cytokine production from mast cells suggests a possible role for normal bacterial DNA in mast cell activation. To examine this issue, we treated mBMMC with DNA preparations from bacterial or control mammalian sources. Whereas E. coli DNA induced both IL-6 and TNF-{alpha} 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).


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  		Table 4. Cytokine Response of mBMMC to Activation with E. coli DNA

 
Neither bacterial DNA nor CpG-ODN induce degranulation of mBMMC
Both bacterial DNA and CpG-ODN have been shown, in our study, to activate mBMMC to secrete cytokines, however, it is also important to know whether these DNA products are capable of degranulating mast cells. We investigated the effects of synthetic ODN on short-term release of ß-hexosaminidase, a well-known marker for mast cell degranulation. mBMMC at 1 x 106 cells/mL were treated for 20 min with either CpG-ODN or control-ODN at 100 µg/mL, the highest dose used in this study that induced substantial levels of cytokine production from mBMMC. Neither CpG-ODN nor control ODN had any significant effects on degranulation by mBMMC as measured by ß-hexosaminidase release after a 20-min activation, whereas calcium ionophore, A23187, a commonly used stimulant for mast cells, induced significant release of this mediator (Fig. 6 ). Examination of degranulation induced by CpG-ODN at a later time point (6 h) also did not suggest any such activity, although the spontaneous release of ß-hexosaminidase was very high under these conditions (spontaneous release 32 ± 12%, CpG-ODN 100 µg/mL 34 ± 9%, n = 4). To examine the effects of bacterial DNA on mBMMC degranulation, mBMMC were treated with 100 µg/mL of E. coli DNA or calf thymus DNA, respectively. Neither of the two bacterial DNA preparations nor the calf thymus DNA induced degranulation from mBMMC. The percentage of ß-hexosaminidase release from mBMMC treated with these natural DNA preparations were 9 ± 1.2, 9 ± 1.5, and 8 ± 0.3%, respectively, mimicking the spontaneous release (10 ± 1.2%) from mast cells incubated with medium alone. The positive control A23187-treated mBMMC had significantly higher levels (21 ± 1.2%) of ß-hexosaminidase release (P < 0.001).



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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.

 
Mast cells can internalize CpG-ODN
The mechanism by which bacterial DNA/CpG-ODN stimulates immune effector cells is presently unknown, but some studies suggest that CpG-ODN render their effects after being taken up by immune cells [3 ]. Understanding how ODN are taken up by different populations of immune effector cells may assist in the design of better methodology to selectively deliver DNA products. This study examined the binding or entering of Texas red-labeled 3CpG-ODN and control-ODN to mBMMC, and demonstrated that both immunostimulatory and control-ODN were bound and taken up by mast cells with similar efficiency.

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.



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  		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

 
To explore the type of pathway primarily involved in internalization of DNA by mast cells, we treated mBMMC for unlabeled-ODN judged by the percentage of positive labeling (84 vs. 71%) and the MFI (27 vs. 25%; Table 4 ).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
During the evolution of immunity, we have developed an efficient innate defense system to quickly recognize and respond to invading pathogens. This is achieved through discrimination of non-self pathogens from self via recognition of certain characteristics or patterns common to infectious agents [32 ]. One example of such a structural pattern is lipopolysaccharide, which is associated with all gram-negative bacteria. According to the danger theory proposed by Matzinger [33 ], LPS and other substances can serve as a signal to host defense systems, resulting in rapid mobilization of innate immune responses. This rapid response to the invading pathogens is usually in the form of acute inflammation.

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-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} production from mBMMC, IL-4, IL-12, IFN-{gamma}, 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-{gamma}, TNF-{alpha}, 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-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} 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-{alpha} 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.


   ACKNOWLEDGEMENTS
 
This work was supported by the Medical Research Council of Canada. We would like thank Mrs. Jing-Yun Shou for providing us with DNA; Drs. Zhi-Qiang Ding and Ping Li for their advice on flow cytometry and confocal microscopy; Mrs. Ursula Kadela-Stolarz and Mrs. Ling Lin for their expert technical assistance; and Ms. Rosa Bailey for her excellent secretarial assistance.

Received June 1, 2000; revised August 14, 2000; accepted August 16, 2000.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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H. Matsushima, N. Yamada, H. Matsue, and S. Shimada
TLR3-, TLR7-, and TLR9-Mediated Production of Proinflammatory Cytokines and Chemokines from Murine Connective Tissue Type Skin-Derived Mast Cells but Not from Bone Marrow-Derived Mast Cells
J. Immunol., July 1, 2004; 173(1): 531 - 541.
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S. Ryzhov, A. E. Goldstein, A. Matafonov, D. Zeng, I. Biaggioni, and I. Feoktistov
Adenosine-Activated Mast Cells Induce IgE Synthesis by B Lymphocytes: An A2B-Mediated Process Involving Th2 Cytokines IL-4 and IL-13 with Implications for Asthma
J. Immunol., June 15, 2004; 172(12): 7726 - 7733.
[Abstract] [Full Text] [PDF]

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R. K. Ikeda, M. Miller, J. Nayar, L. Walker, J. Y. Cho, K. McElwain, S. McElwain, E. Raz, and D. H. Broide
Accumulation of Peribronchial Mast Cells in a Mouse Model of Ovalbumin Allergen Induced Chronic Airway Inflammation: Modulation by Immunostimulatory DNA Sequences
J. Immunol., November 1, 2003; 171(9): 4860 - 4867.
[Abstract] [Full Text] [PDF]

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