Asthma is an inflammatory disease of the airways that is induced by Th2 cytokines and inhibited by Th1 cytokines. Despite a steady increase in the incidence, morbidity, and mortality from asthma, no current treatment can reduce or prevent asthma for a prolonged period. We examined the ability of unmethylated CpG oligodeoxynucleotides (ODN), which are potent inducers of Th1 cytokines, to prevent the inflammatory and physiological manifestations of asthma in mice sensitized to ragweed allergen. Administration of CpG ODN 48 h before allergen challenge increased the ratio of IFN-γ to IL-4 secreting cells, diminished allergen-induced eosinophil recruitment, and decreased the number of ragweed allergen-specific IgE-producing cells. These effects of CpG ODN were sustained for at least 6 wk after its administration. Furthermore, there was a vigorous Th1 memory response to the recall Ag, inhibition of peribronchial and perivascular lung inflammation, and inhibition of bronchial hyperresponsiveness 6 wk after administration of CpG ODN. Administration of CpG ODN in IFN-γ −/− mice failed to inhibit eosinophil recruitment, indicating a critical role of IFN-γ in mediating these effects. This is the first report of a treatment that inhibits allergic lung inflammation in presensitized animals for a prolonged period and thus has relevance to the development of an effective long term treatment for asthma.

Asthma is an inflammatory disease of the airways that affects 5–10% of the general population. Epidemiological studies indicate that there is a global increase in the incidence, morbidity, and mortality caused by asthma despite an expanding repertoire of medications available for the treatment of this disease (1, 2). These observations underscore the need for therapeutic strategies that more effectively prevent asthma.

Airway eosinophilia, bronchial hyperresponsiveness, and increased levels of allergen-specific IgE are characteristic of asthma. Thus, an effective long term treatment for asthma should inhibit one or more of these processes. Considerable evidence indicates that eosinophils play a key role in mediating injury to the bronchial mucosa (3, 4, 5). In addition to airway epithelial injury, eosinophil granule proteins increase airway reactivity to acetylcholine in vitro and in vivo (5, 6, 7). During seasonal allergen exposure, there is an increase in ragweed allergen (RW)-specific3 serum IgE levels to an extent that it can account for 50% of total serum IgE (8). Evidence suggests an important role for allergen-specific IgE in eosinophil recruitment during the allergic late phase inflammatory response (9, 10). Th2 cytokines are important in both the recruitment and activation of lung eosinophils and production of IgE (11, 12). The number of cells expressing Th2 cytokine mRNA are increased during allergic inflammation, and depletion of CD4+ cells in vivo attenuates allergen-induced eosinophilic lung inflammation (12, 13). IL-4 knockout mice and animals treated with anti-IL-4 demonstrate reduced allergen-induced eosinophil recruitment (14, 15). In contrast, we and others have shown that Th1 cytokines such as IFN-γ and IL-12 inhibit the development of allergic lung inflammation (16, 17). Thus, agents that selectively elicit a prolonged Th1 immunity might be useful in inhibiting allergic lung inflammation in asthma.

Our laboratory has been examining the immunomodulatory activity of synthetic oligodeoxynucleotides (ODN) expressing CpG motifs that consist of a central unmethylated CpG dinucleotide flanked by two 5′-purines and two 3′-pyrimidines. We and others found that CpG ODN rapidly stimulate T, B, NK, and macrophages to proliferate, secrete Abs, and/or produce a variety of Th1-associated cytokines, predominantly IFN-γ and IL-12 (18, 19). Kline et al. (20) demonstrated that systemically administered CpG ODNs could reduce the allergic response of mice sensitized and challenged with Schistosoma eggs. In that study, CpG ODN was administered i.p., and their effect was monitored for 2 wk. Broide et al. (21) recently reported that administration of CpG ODN inhibits allergic responses in mice sensitized and challenged with OVA. In that study, CpG ODNs were administered i.p., intranasally, or intratracheally, and their effects were monitored for 1–6 days.

In this study, we examined whether intratracheal administration of CpG ODN (modeling the effect of nebulizer delivery to humans) could alter the immunological and physiological manifestations of ragweed-induced asthma in mice. These experiments used animals that were presensitized by allergen to mount a pathological Th2 allergic response. We found that CpG ODN administered 2 days before RW challenge converted the predominantly Th2 allergic response to a dominant Th1 response and significantly reduced lung eosinophilia and RW-specific IgE production. The beneficial effects lasted for at least 6 wk after the last dose of CpG ODN, indicating a prolonged effect.

Female BALB/c mice, 6–8 wk old, were purchased from the Harlan Laboratories (Indianapolis, IN) to perform all experiments except those requiring IFN-γ −/− and IFN-γ +/+ mice. The latter (IFN-γ −/− and IFN-γ +/+ mice) were 5-wk-old female BALB/c mice that were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice were maintained in a specific pathogen-free environment throughout the experiment.

Two immunostimulatory unmethylated CpG-containing ODNs of sequence GCTAGACGTTAGCGT and TCAACGTT were synthesized as described (18, 19). Control ODN were synthesized by eliminating the CpG motifs by inversion (GCTAGAGCTTAGGCT, TCAAGCTT) or by methylating the cytosine residues in the CpG motifs. All ODNs were produced on the same synthesizer and were purified by extraction with phenol-chloroform-isoamyl alcohol (25:24:1) followed by ethanol precipitation. These ODN contained undetectable levels of endotoxin (<0.02 U/kg, as determined using a Limulus amebocyte lysate analysis kit (QCL-1000 BioWhittaker, Walkersville, MD). All ODN were administered at a dose of 35 μg/100 μl/intratracheal instillation.

Endotoxin-free ragweed (lot XP56-D10-1320) was purchased from Greer Laboratories (Lenoir, NC). All experiments were performed with ragweed because it is an allergen relevant to human allergic asthma. We previously showed that patients with allergic asthma challenged subsegmentally with RW and other allergens mount a late phase airway inflammation that is either predominantly neutrophilic or eosinophilic, depending on the quantity of endotoxin in the allergenic extract (22). Because asthma is an eosinophilic disease of the airways, we used endotoxin-free ragweed extract in the current study.

Two models were used to evaluate the effects of CpG ODN on ragweed asthma, the short term model and the long term model (Tables I and II). In the short term model, BALB/c mice were sensitized by i.p. injection of 150 μg of RW plus alum on days 0 and 4, as described (17). ODN (35 μg/100 μl/mouse) were administered intratracheally 0–48 h before allergen challenge (200 μg of RW administered intratracheally), which was performed on day 11. Mice were sacrificed and studied on day 14 for bronchoalveolar lavage (BAL) cell counts. In additional animals, the lungs and spleen were dissected for enzyme-linked immunospot (ELISPOT) cytokine analysis 3 days after the final RW challenge.

In the long term model (Table II), mice were sensitized with RW as described above and challenged with RW intratracheally on days 11, 25, and 65 (to mimic repeated seasonal allergen exposure). Groups of animals were treated with PBS or CpG ODN 2 days before each allergen challenge. A control group (RW/PBS) was treated with PBS on days 9, 23, and 63. To evaluate the long term effects of CpG ODN, the second group (RW/CpG-6 wk) received 35 μg of CpG ODN intratracheally on days 9 and 23 and PBS on day 63. A third treatment group (RW/CpG-2 days) received CpG ODN 2 days before each of the three allergen challenges. This group was included to test whether repeated doses of CpG ODN before each of the ragweed challenges was required to maintain its activity. The third group in the long term asthma model was similar to the RW/CpG-48 h group in the short term model in that the last dose of CpG ODN was administered 48 h before the final allergen challenge.

Table II.

Long term asthma modela

Day 9Day 11Day 23Day 25Day 63Day 65Day 68
RW/PBS PBS i.t. RW i.t. PBS i.t. RW i.t. PBS i.t. RW i.t. BAL, ELISPOT etc. 
RW/CpG-6 wk CpG i.t. RW i.t. CpG i.t. RW i.t. PBS i.t. RW i.t. BAL, ELISPOT etc. 
RW/CpG-2 days CpG i.t. RW i.t. CpG i.t. RW i.t. CpG i.t. RW i.t. BAL, ELISPOT etc. 
Day 9Day 11Day 23Day 25Day 63Day 65Day 68
RW/PBS PBS i.t. RW i.t. PBS i.t. RW i.t. PBS i.t. RW i.t. BAL, ELISPOT etc. 
RW/CpG-6 wk CpG i.t. RW i.t. CpG i.t. RW i.t. PBS i.t. RW i.t. BAL, ELISPOT etc. 
RW/CpG-2 days CpG i.t. RW i.t. CpG i.t. RW i.t. CpG i.t. RW i.t. BAL, ELISPOT etc. 
a

Mice were sensitized with two doses of 150 μg of RW + alum i.p. given on days 0 and 4. In the RW/CpG-15 h group, CpG ODN was given 15 h before allergen challenge. In the RW/CpG-48 h group, CpG ODN was given 48 h before allergen challenge. i.t., intratracheal; CpG i.t., i.t. CpG ODN 35 μg; RW i.t., i.t. 200 μg of ragweed.

In the long term model, pulmonary function testing was performed with the Buxco system on day 67, 2 days after the final RW challenge. Because pulmonary function testing was performed in unrestrained, conscious animals (to mimic methacholine challenge in patients with asthma), the same animals could be used to collect BAL and to perform histological analysis the following day. In additional animals, the lungs and spleen were dissected for ELISPOT cytokine and Ig analysis, and serum was collected for Ig analysis by ELISA 3 days after the final RW challenge.

IFN-γ +/+ and IFN-γ −/− female BALB/c mice, 5 wk old, were purchased from The Jackson Laboratory. A protocol identical with that for the RW/CpG-48 h group in the short term model (Table I) was performed. Seventy-two hours after RW challenge, the mice underwent BAL, and the collected BAL fluid was analyzed for total and differential immune cell counts.

Table I.

Short term asthma modela

Day 9Day 10Day 11Day 14
RW/PBS PBS i.t. PBS i.t. RW+ PBS i.t. BAL, ELISPOT 
RW/CpG-48 h CpG i.t. PBS i.t. RW+ PBS i.t. BAL, ELISPOT 
RW/CpG-15 h PBS i.t. CpG i.t. RW+ PBS i.t. BAL, ELISPOT 
RW/CpG 0 h PBS i.t. PBS i.t. RW+ CpG i.t. BAL, ELISPOT 
RW/GpC-48 h GpC i.t. PBS i.t. RW+ PBS i.t. BAL, ELISPOT 
RW/mCpG-48 h mCpG i.t. PBS i.t. RW+ PBS i.t. BAL, ELISPOT 
Day 9Day 10Day 11Day 14
RW/PBS PBS i.t. PBS i.t. RW+ PBS i.t. BAL, ELISPOT 
RW/CpG-48 h CpG i.t. PBS i.t. RW+ PBS i.t. BAL, ELISPOT 
RW/CpG-15 h PBS i.t. CpG i.t. RW+ PBS i.t. BAL, ELISPOT 
RW/CpG 0 h PBS i.t. PBS i.t. RW+ CpG i.t. BAL, ELISPOT 
RW/GpC-48 h GpC i.t. PBS i.t. RW+ PBS i.t. BAL, ELISPOT 
RW/mCpG-48 h mCpG i.t. PBS i.t. RW+ PBS i.t. BAL, ELISPOT 
a

Mice were sensitized with two doses of 150 μg of RW + alum i.p. given on days 0 and 4. In the RW/CpG-15 h group, CpG ODN was given 15 h before allergen challenge. In the RW/CpG-48 h group, CpG ODN was given 48 h before allergen challenge. i.t., intratracheal; CpG i.t., i.t. CpG ODN 35 μg; RW i.t., i.t. 200 μg of ragweed.

Mice were euthanized with an i.p. injection of ketamine and xylazine to perform BAL as previously described (17). BAL fluids were obtained by cannulating the trachea and lavaging the lungs with two 0.7-ml aliquots of ice cold Dulbecco’s PBS (Sigma Chemical, St. Louis, MO). The BAL cells were pelleted, washed, and stained with Wright-Giemsa. The number of eosinophils, neutrophils, lymphocytes, and macrophages was determined by microscopic examination of a minimum of 200 cells/slide of a cytocentrifuge preparation.

ELISPOT assays and serum IgE assays were performed in parallel experiments as described below. Animals were bled by retroorbital puncture, and serum stored at −20°C until use. Mice were killed by cervical dislocation, and their spleens and lungs were removed aseptically. Mouse spleens and lungs were minced and passed through a wire mesh to generate single-cell suspensions. Where indicated, cells were incubated in complete media (RPMI 1640 plus 10% FBS, 1.5 mM l-glutamine, penicillin, and streptomycin at 100 units/ml).

Splenocytes were incubated at 5 × 106 cells/ml with either diluent or 100 μg/ml ragweed for 4 days. The cells were incubated in complete medium at 37°C in a humidified 5% CO2 incubator. The cell supernatants were examined for IFN-γ levels with a two-site immunoenzymetric assay using anti-IFN-γ Abs (clones R4-6A2 and XMG1.2, PharMingen, San Diego, CA).

Immulon 2 microtiter plates (96-well) were coated with 10 μg/ml anti-IFN-γ (clone RA6a2, Lee Biomolecular, San Diego, CA) or anti-IL-4 (clone BVD4-1D11, Endogen, Woburn, MA) in 0.1 M carbonate buffer (pH 9.6) for 3 h at room temperature (19). The plates were blocked with PBS-5% BSA for 1 h and washed with PBS-0.025% Tween 20. Serial dilutions of single spleen or lung cell suspensions, ranging from 1 to 10 × 105 cells/well, were incubated on anti-cytokine-coated plates in complete medium for 8–10 h at 37°C in a humidified 5% CO2 incubator. Plates were then washed with PBS-Tween 20 and overlaid with 1 μg/ml biotinylated anti-IFN-γ (clone XMG 1.2, PharMingen) or anti-IL-4 (clone BVD6-24G2, Endogen), washed, and treated with a 1:2000 dilution of avidin-conjugated alkaline phosphatase (Vector Laboratories, Burlingame, CA) for 2 h at room temperature. After a final wash, the cytokine products of individual secreting cells were visualized by the addition of a solution of 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (Kirkegaard and Perry, Gaithersburg, MD). ELISPOTs were counted with the aid of a dissecting microscope and expressed as ELISPOTs per million cells.

Immulon 1 microtiter plates (96 wells) were coated with 10 μg/ml ragweed protein and blocked for 1 h with PBS-1% BSA. Plates were overlaid with serially diluted cells or sera, incubated as described above, washed, and reacted with phosphatase-conjugated anti-mouse IgG1, IgG2a (Southern Biotechnology, Birmingham, AL) or IgE (a generous gift from Dr. Clifford Snapper). Serum Ab concentrations were determined by comparison to a serially diluted high titered positive control, while ELISPOTS were quantitated as described above.

The ELISPOT data were used to calculate the ratio of IFN-γ-IL-4-secreting cells. The fold increases in the ratio of IFN-γ to IL-4 cells for each experimental group were determined by comparing it to the same ratio in PBS-treated controls. The spleen and lung IFN-γ-IL-4 ratios were calculated separately.

Airway responsiveness was measured in unrestrained animals using whole body plethysmography (BUXCO, Troy, NY) as described by Hamelmann et al. (23), with minor revisions. Mice were placed in the main chamber of the plethysmograph, and baseline readings were taken and averaged for 5 min. In a separate chamber, mice were exposed for 3-min intervals to nebulized PBS followed by 3–50 mg/ml methacholine administered with a Pari LC star turbo nebulizer (Pari Respiratory Equipment, Midlothian, VA). Mice were returned to the plethysmograph chamber, and airway responsiveness was analyzed for 5 min following the PBS exposure and exposure to each dose of methacholine. Airway reactivity was expressed as a fold increase in enhanced pause (PENH) for each concentration of methacholine relative to the PENH values produced by PBS exposure for individual mice (PENH index).

Following BAL, the lungs were infused with 1 ml of 10% neutral buffered formalin solution. The fixed lungs were embedded in paraffin, sectioned at a thickness of 4 μm, and stained with hematoxylin and eosin. A random number was assigned to each hematoxylin and eosin-stained lung section from the treatment groups. A pathologist blinded to the random numbers evaluated the slides for the degree of inflammation using a Zeiss photomicroscope (Zeiss, Oberkochen, Germany). The degree of peribronchial and perivascular inflammation was evaluated on a subjective scale of 0, 1, 2, 3, and 4 corresponding to none, mild, moderate, marked, or severe inflammation, respectively, with an increment of 0.5 if the inflammation fell between two integers (17, 24). The total lung inflammation was defined as the sum of peribronchial and perivascular inflammation scores.

The difference in BAL cell counts between treatment groups was analyzed by one-way ANOVA. Significant ANOVAs were further analyzed by the Bonnferroni/Dunns post hoc test. All ELISPOT results were analyzed by one-way ANOVA using SigmaStat (Jandel Scientific, San Rafael, CA), and significant ANOVAs were checked by Student-Newman-Keuls post hoc test to establish normality and significance. The limited number of lung cells that could be derived from each animal required performing ELISPOT assays on pooled lung cells from 6 animals/group. Thus, statistically significant differences between groups cannot be established for these samples. The comparison of histology scores was analyzed by the Mann-Whitney test. The PENH data were analyzed by Student’s t test.

When BALB/c mice were sensitized and challenged with RW, they rapidly developed the immunological abnormalities characteristic of an allergic response. Thus, the total number of BAL cells in the RW/PBS group rose 5-fold (p < 0.0001), whereas the total number of eosinophils rose 400-fold (p < 0.0001) when compared with naive animals (Fig. 1). We examined whether the timing of intratracheal administration of CpG ODN with respect to allergen challenge influenced the development of allergic lung inflammation. Compared with the RW/PBS group, administration of CpG ODN 48 and 15 h before RW challenge inhibited BAL eosinophil numbers by 70% (p < 0.0001) and 55% (Fig. 1,A, p < 0.001), respectively. Administration of CpG ODN 48 h before RW challenge also reduced total immune cell counts in the BAL fluids by 50% (Fig. 1 B, p < 0.0001). In contrast, total BAL immune cell counts and eosinophil numbers were not significantly reduced by the administration of negative control ODN in which the critical CpG dinucleotide was inverted or the cytosine bases were methylated (RW/GpC-48 h and RW/mCpG-48 h groups, respectively).

FIGURE 1.

Rapid effects of CpG ODN on eosinophil and total BAL cell recruitment. BALB/c mice (Harlan) were sensitized with two doses of ragweed (RW) and alum on days 0 and 4 and challenged intratracheally with RW on day 11. CpG ODN was administered intratracheally simultaneously with (RW/CpG 0 h, n = 8), 15 h before (RW/CpG-15 h, n = 9) or 48 h before (RW/CpG-48 h, n = 11) RW challenge. Three control groups received either intratracheal PBS (RW/PBS, n = 18) or a control ODN in which the critical CpG motif was reversed to GpC (RW/GpC-48 h, n = 9), or methylated CpG ODN (RW/mCpG-48 h, n = 4) 48 h before RW challenge. Three days after RW challenge BALs were performed on each group of mice, and total and differential BAL immune cell counts were determined. A, BAL eosinophil cell numbers × 104/ml in the different groups; B, BAL total cells × 104/ml in the different groups. Values are expressed as mean ± SEM. ++++, p < 0.0001 compared with naive animals; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001 compared with RW/PBS treated group.

FIGURE 1.

Rapid effects of CpG ODN on eosinophil and total BAL cell recruitment. BALB/c mice (Harlan) were sensitized with two doses of ragweed (RW) and alum on days 0 and 4 and challenged intratracheally with RW on day 11. CpG ODN was administered intratracheally simultaneously with (RW/CpG 0 h, n = 8), 15 h before (RW/CpG-15 h, n = 9) or 48 h before (RW/CpG-48 h, n = 11) RW challenge. Three control groups received either intratracheal PBS (RW/PBS, n = 18) or a control ODN in which the critical CpG motif was reversed to GpC (RW/GpC-48 h, n = 9), or methylated CpG ODN (RW/mCpG-48 h, n = 4) 48 h before RW challenge. Three days after RW challenge BALs were performed on each group of mice, and total and differential BAL immune cell counts were determined. A, BAL eosinophil cell numbers × 104/ml in the different groups; B, BAL total cells × 104/ml in the different groups. Values are expressed as mean ± SEM. ++++, p < 0.0001 compared with naive animals; ∗∗∗, p < 0.001; ∗∗∗∗, p < 0.0001 compared with RW/PBS treated group.

Close modal

Compared with naive animals, there was a 9-fold increase in IgE producing spleen cells (p < 0.05) and >18-fold increase in IgE-producing lung cells (Table III) in allergen-sensitized, PBS-treated animals. Administration of CpG ODN 48 h before allergen challenge reduced the number of IgE-secreting cells in the spleen by 44% (p < 0.05) and the lungs by 83%. These results indicate that CpG ODN rapidly inhibits eosinophilic lung inflammation and IgE production in a ragweed model of allergic asthma.

Table III.

Rapid effects of CpG ODN on IgE-producing cells in the spleen and lunga

RW-Specific IgE-Secreting Cells/Million
SpleenLung
Naive 2 ± 0.6 
RW/PBS 18 ± 1.2+ 20 
RW/CpG-48 h 10 ± 1.2b 
RW/GpC-48 h 24 ± 2.3 18 
RW-Specific IgE-Secreting Cells/Million
SpleenLung
Naive 2 ± 0.6 
RW/PBS 18 ± 1.2+ 20 
RW/CpG-48 h 10 ± 1.2b 
RW/GpC-48 h 24 ± 2.3 18 
a

Mice were sacrificed 3 days after RW challenge, and RW-specific IgE ELISPOT assays were performed on single-cell suspensions of spleen and lung cells. Spleen values are expressed as the mean ± SEM for three animals. Because only a limited number of lung cells could be derived from each animal, the lung values represent the ELISPOT results of pooled cells from three animals.

+, p < 0.05 compared with naive animals.

b

p < 0.05 compared with the RW/PBS group.

Cytokine ELISPOT assays were used to monitor the number of cells actively secreting IFN-γ and IL-4 in the lungs and spleen of mice sensitized and challenged with RW. The number of spleen cells producing either of these cytokines was significantly higher in the RW/PBS-treated mice than in naive controls (p < 0.05; Fig. 2,A). When administered 48 h before allergen challenge, CpG ODN significantly increased the number of IFN-γ producing cells in the spleen (p < 0.05; Fig. 2,A). This altered the cytokine milieu by increasing the ratio of IFN-γ-IL-4-secreting cells 3.5-fold when compared with the RW/PBS group. This effect required administration of the CpG motif, because no change was observed in the RW/GpC-48 h group. Similarly, administration of CpG ODN 48 h before allergen challenge increased the number of IFN-γ-producing cells in the lungs (Fig. 2 B) and increased the ratio of IFN-γ-IL-4-secreting cells 3.5-fold relative to the RW/PBS group. Thus, intratracheal administration of CpG ODN in the short term asthma model increases the number of cells producing Th1 cytokines in the lungs and systemically in the spleen.

FIGURE 2.

Rapid effects of CpG ODN on IFN-γ- and IL-4-secreting cells in the lungs and spleen. ELISPOT assays were performed on single-cell suspensions of spleen and lung cells obtained from the animal groups described in Fig. 1. Shown are the number of IFN-γ- and IL-4-secreting cells per million spleen (A) or lung (B) cells. For A (spleen), each bar represents the mean ± SEM values for three animals. Because only a limited number of lung cells could be derived from each animal, each bar in B (lung) represents the ELISPOT results of pooled lung cells from three animals. +, p < 0.05 compared with naive animals; ∗, p < 0.05 compared with the RW/PBS group.

FIGURE 2.

Rapid effects of CpG ODN on IFN-γ- and IL-4-secreting cells in the lungs and spleen. ELISPOT assays were performed on single-cell suspensions of spleen and lung cells obtained from the animal groups described in Fig. 1. Shown are the number of IFN-γ- and IL-4-secreting cells per million spleen (A) or lung (B) cells. For A (spleen), each bar represents the mean ± SEM values for three animals. Because only a limited number of lung cells could be derived from each animal, each bar in B (lung) represents the ELISPOT results of pooled lung cells from three animals. +, p < 0.05 compared with naive animals; ∗, p < 0.05 compared with the RW/PBS group.

Close modal

We next examined the duration of CpG ODN activity by utilizing a long term asthma model. This model produces a greater magnitude of lung inflammation than the short term model because each group received three intratracheal ragweed challenges over an 8-wk period (mimicking repeated seasonal allergen exposure). The RW/PBS and RW/CpG-2 days groups received three doses of PBS or CpG ODN, respectively, by intratracheal administration 2 days before each of the three subsequent RW challenges. The RW/CpG-6 wk group was designed to examine the long term effects of CpG ODN and was administered CpG ODN before the first two RW challenges but not before the final RW challenge 6 wk later.

Compared with naive animals, repeated challenge with RW resulted in a substantial rise in the number of cells secreting IL-4 in both lungs and spleen (Fig. 3, A and B). Consistent with results from the short term model, treating mice with CpG ODN 2 days before all of the RW challenges (RW/CpG-2 days group) increased the ratio of IFN-γ-IL-4-secreting cells in the lungs and spleen by 4–5-fold. Among mice treated with CpG ODN and then challenged 6 wk later with RW (RW/CpG-6 wk group), the ratio of IFN-γ-IL-4-secreting cells in the lungs increased 3–4-fold when compared with the RW/PBS group. In contrast, there was no difference in this ratio among spleen cells from this group compared with the RW/PBS group. These results suggest that RW-specific Th1 cells are localized in the lungs 6 wk after intratracheal administration of CpG ODN.

FIGURE 3.

Long term effects of CpG ODN on IFN-γ- and IL-4-secreting cells in the lungs and spleen and on spleen recall response. Three groups of BALB/c mice were sensitized with two i.p. doses of ragweed and alum. A control group (RW/PBS) was treated with PBS on days 9, 23, and 63. A second group (RW/CpG-2 days) received three doses of CpG on days 9, 23, and 63. To evaluate the long term effects of CpG, one group (RW/CpG-6 wk) received 35 μg of CpG intratracheally on days 9 and 23 and PBS on day 63. All three groups were challenged intratracheally with RW (200 μg/100 μl) on days 11, 25, and 65. Three days after RW challenge, the animals were euthanized to perform ELISPOT assays for IL-4 and IFN-γ. Shown are the number of IFN-γ- and IL-4-secreting cells per million spleen (A) or lung (B) cells. For A (spleen), each bar represents the mean ± SEM value for six animals. Because only a limited number of lung cells could be derived from each animal, each bar in B (lung) represents the ELISPOT results of pooled lung cells from 6 animals. +, p < 0.05 compared with naive animals; ∗, p < 0.05 compared with the RW/PBS group. For recall experiments, mice from the different groups of animals described in the long-term protocol were sacrificed 3 days after RW challenge. Splenocytes from animals in the RW/PBS group (n = 6), RW/CpG-6 wk group (n = 6) and RW/CpG-2 days group (n = 3) were stimulated in in vitro for 4 days with vehicle or RW. After 4 days of culture, the cell supernatants were analyzed for IFN-γ levels by ELISA. C, Each column shows the mean ± SEM for recall production of IFN-γ, which is defined as the difference between IFN-γ produced by splenocytes incubated with RW and those incubated with vehicle control. ∗∗, p < 0.01 and ∗∗∗, p < 0.001 compared with the corresponding RW/PBS group.

FIGURE 3.

Long term effects of CpG ODN on IFN-γ- and IL-4-secreting cells in the lungs and spleen and on spleen recall response. Three groups of BALB/c mice were sensitized with two i.p. doses of ragweed and alum. A control group (RW/PBS) was treated with PBS on days 9, 23, and 63. A second group (RW/CpG-2 days) received three doses of CpG on days 9, 23, and 63. To evaluate the long term effects of CpG, one group (RW/CpG-6 wk) received 35 μg of CpG intratracheally on days 9 and 23 and PBS on day 63. All three groups were challenged intratracheally with RW (200 μg/100 μl) on days 11, 25, and 65. Three days after RW challenge, the animals were euthanized to perform ELISPOT assays for IL-4 and IFN-γ. Shown are the number of IFN-γ- and IL-4-secreting cells per million spleen (A) or lung (B) cells. For A (spleen), each bar represents the mean ± SEM value for six animals. Because only a limited number of lung cells could be derived from each animal, each bar in B (lung) represents the ELISPOT results of pooled lung cells from 6 animals. +, p < 0.05 compared with naive animals; ∗, p < 0.05 compared with the RW/PBS group. For recall experiments, mice from the different groups of animals described in the long-term protocol were sacrificed 3 days after RW challenge. Splenocytes from animals in the RW/PBS group (n = 6), RW/CpG-6 wk group (n = 6) and RW/CpG-2 days group (n = 3) were stimulated in in vitro for 4 days with vehicle or RW. After 4 days of culture, the cell supernatants were analyzed for IFN-γ levels by ELISA. C, Each column shows the mean ± SEM for recall production of IFN-γ, which is defined as the difference between IFN-γ produced by splenocytes incubated with RW and those incubated with vehicle control. ∗∗, p < 0.01 and ∗∗∗, p < 0.001 compared with the corresponding RW/PBS group.

Close modal

To identify RW-specific Th1 memory cells, splenocytes from all three groups were restimulated in vitro with the recall Ag (RW). As seen in Fig. 3 C, splenocytes derived from both the RW/CpG-2 days and RW/CpG-6 wk groups responded vigorously to the recall Ag with IFN-γ production. Compared with the RW/PBS group, the RW/CpG-6 wk group demonstrated a 4.7-fold increase (p < 0.001) and the RW/CpG-2 days group demonstrated a 5.5-fold increased (p < 0.01) production of IFN-γ. Thus, in addition to increasing the ratio of IFN-γ-IL-4-secreting cells, intratracheal administration of CpG-ODN stimulated an Ag-specific Th1 memory response that persisted for at least 6 wk.

We then evaluated the production of anti-RW Abs between treatment groups. Repeated challenge with RW (RW/PBS group) resulted in a 17-fold elevation in serum ragweed-specific IgE levels compared with naive animals (Fig. 4,A). Compared with RW/PBS group, the fold increase in serum ragweed-specific IgE was 73% lower in the RW/CpG-2 days group (p < 0.05) and 43% lower in the RW/CpG-6 wk group (p < 0.05). The spleen and the lungs had similar numbers (46 and 57 secreting cells per million) of ragweed-specific IgE secreting cells in the RW/PBS treated animals (Fig. 4B). Compared with PBS treated animals (RW/PBS group), the RW/CpG-2 days group had 65% fewer RW-specific IgE producing cells in the spleen (p < 0.05) and 68% fewer in the lungs. In the RW/CpG-6 wk group, there were 17% fewer cells in the spleen, and 39% fewer in the lungs. These findings suggest that the increased ratio of IFN-γ-IL-4 secreting cells resulting from CpG ODN administration may have altered the milieu in which anti-RW Ig secreting B-cells mature, and this effect was longer lasting in the lungs than spleen.

FIGURE 4.

Long term effects of CpG ODN on ragweed-specific Ab production. Mice from the different groups of animals described in Fig. 3 were sacrificed 3 days after RW challenge. Serum RW-specific IgE was measured by ELISA, and the number of spleen and lung B cells secreting ragweed-specific IgE, IgG1, and IgG2a were measured by ELISPOT assay. A, Fold increase in RW-specific serum IgE for the groups in the long term protocol compared with naive animals. Each bar in the histogram represents the mean ± SEM for six animals. BD, Isotype-specific anti-RW-secreting cells in the spleen and lungs. The group designations shown in B also apply to C and D. For spleen, the data represent mean ± SEM for six animals. Because only a limited number of lung cells could be derived from each animal, each bar for the lung data represents the ELISPOT results of pooled lung cells from six animals. +, p < 0.05 compared with naive animals; ∗, p < 0.05 compared with the RW/PBS group.

FIGURE 4.

Long term effects of CpG ODN on ragweed-specific Ab production. Mice from the different groups of animals described in Fig. 3 were sacrificed 3 days after RW challenge. Serum RW-specific IgE was measured by ELISA, and the number of spleen and lung B cells secreting ragweed-specific IgE, IgG1, and IgG2a were measured by ELISPOT assay. A, Fold increase in RW-specific serum IgE for the groups in the long term protocol compared with naive animals. Each bar in the histogram represents the mean ± SEM for six animals. BD, Isotype-specific anti-RW-secreting cells in the spleen and lungs. The group designations shown in B also apply to C and D. For spleen, the data represent mean ± SEM for six animals. Because only a limited number of lung cells could be derived from each animal, each bar for the lung data represents the ELISPOT results of pooled lung cells from six animals. +, p < 0.05 compared with naive animals; ∗, p < 0.05 compared with the RW/PBS group.

Close modal

To pursue this observation, we analyzed the frequency of B cells secreting RW-specific IgG1 and IgG2a. Consistent with the ability of IFN-γ to induce isotype switching from IgG1 to IgG2a, CpG ODN decreased the number of IgG1-producing cells (Fig. 4,C) and increased the number of IgG2a-secreting cells in the lungs (Fig. 4 D). Compared with PBS-treated animals (RW/PBS group), the number of IgG1-secreting cells in the RW/CpG-2 days group were 77% fewer in the spleen (p < 0.05) and 38% fewer in the lungs. Similarly, in the RW/CpG-6 wk group, there were 57% fewer cells in the spleen (p < 0.05) and 36% fewer in the lungs. Compared with the RW/PBS group, the RW/CpG-2 days group and the RW/CpG-6 wk group demonstrated 6.3-fold and 5-fold more IgG2a-producing lung cells, respectively. Although far fewer IgG2a-producing cells were present in the spleen, a similar pattern of effects of CpG ODN administration on IgG2a secretion was observed in this organ. The RW/CpG-2 days and RW/CpG-6 wk groups demonstrated significantly more IgG2a-producing splenocytes compared with the RW/PBS group (p < 0.05). These results provide further evidence that the increased ratio of IFN-γ-IL-4 secreting cells resulting from CpG ODN administration altered the milieu for the maturation of B cells secreting anti-RW Igs from a Th2 bias to a Th1 bias.

We sought to determine whether CpG ODN have long term antiallergic effects. In the RW/CpG-6 wk group, the BAL eosinophil and total immune cell counts were inhibited 66% (p < 0.01) and 48% (p < 0.01), respectively (Fig. 5, A and B) compared with the RW/PBS group. Similarly, in the RW/CpG-2 days group, the BAL eosinophil and total immune cell recruitment were inhibited 76% (p < 0.001) and 53% (p < 0.01), respectively. These results were similar in magnitude to the inhibition of BAL eosinophils produced by CpG ODN administered 48 h before challenge (70%) in the short term asthma model described above. Furthermore, there was no significant difference between the reduction in eosinophils and total cells demonstrated by the RW/CpG-2 days and the RW/CpG-6 wk groups, indicating that the inhibitory effects of CpG ODN on allergic lung inflammation are sustained for at least 6 wk.

FIGURE 5.

Long term effects of CpG ODN on BAL eosinophil and total immune cell recruitment. Mice from the different groups of animals identified in Fig. 4 were sacrificed 3 days after RW challenge. A, BAL eosinophil cell numbers × 104/ml in the different groups. B, BAL total cells × 104/ml in the different groups. Values are expressed as mean ± SEM for six or seven animals. ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared with RW/PBS-treated group.

FIGURE 5.

Long term effects of CpG ODN on BAL eosinophil and total immune cell recruitment. Mice from the different groups of animals identified in Fig. 4 were sacrificed 3 days after RW challenge. A, BAL eosinophil cell numbers × 104/ml in the different groups. B, BAL total cells × 104/ml in the different groups. Values are expressed as mean ± SEM for six or seven animals. ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared with RW/PBS-treated group.

Close modal

Following BAL, the lungs were formalin fixed, embedded in paraffin, sectioned at 4 μm thickness, and stained with hematoxylin and eosin. Total lung inflammation was determined histologically and defined as the sum of peribronchial and perivascular inflammation scores. Fig. 6,A (naive), 6B (RW/PBS) and 6C (RW/CpG-6 wk) show representative hematoxylin and eosin-stained peribronchial and perivascular areas of the lungs (×40). Naive animals had no detectable lung inflammation, whereas the RW/PBS group had significant peribronchial and perivascular inflammation. Compared with the RW/PBS group, the peribronchial (56% inhibition, p < 0.01), the perivascular (47% inhibition, p < 0.01), and the total (51% inhibition, p < 0.001) lung inflammation were inhibited in the RW/CpG-6 wk group (Table IV).

FIGURE 6.

Long term effects of CpG ODN on peribronchial and perivascular lung inflammation. The lungs of a naive group and the RW/PBS and RW/CpG-6 wk long term groups were fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. A pathologist blinded to the treatment groups evaluated the degree of peribronchial and perivascular inflammation on a scale of 0 to 4 with an increment of 0.5 if the inflammation fell between two integers. The total lung inflammation was defined as the sum of peribronchial and perivascular inflammation scores. Shown are photographs of lung sections from representative animals within each group at ×40 magnification. Open curved arrows show a bronchus, and closed straight arrows show a vessel. The lung of a naive animal (A) shows no peribronchial or perivascular inflammation. In contrast, the lung of an animal from the RW/PBS group (B) shows grade 4 peribronchial inflammation, grade 4 perivascular inflammation, and grade 8 total lung inflammation. The lung from an animal in the RW/CpG-6 wk group (C) shows grade 1 peribronchial inflammation, grade 1.5 perivascular inflammation, and grade 2.5 total lung inflammation.

FIGURE 6.

Long term effects of CpG ODN on peribronchial and perivascular lung inflammation. The lungs of a naive group and the RW/PBS and RW/CpG-6 wk long term groups were fixed in formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. A pathologist blinded to the treatment groups evaluated the degree of peribronchial and perivascular inflammation on a scale of 0 to 4 with an increment of 0.5 if the inflammation fell between two integers. The total lung inflammation was defined as the sum of peribronchial and perivascular inflammation scores. Shown are photographs of lung sections from representative animals within each group at ×40 magnification. Open curved arrows show a bronchus, and closed straight arrows show a vessel. The lung of a naive animal (A) shows no peribronchial or perivascular inflammation. In contrast, the lung of an animal from the RW/PBS group (B) shows grade 4 peribronchial inflammation, grade 4 perivascular inflammation, and grade 8 total lung inflammation. The lung from an animal in the RW/CpG-6 wk group (C) shows grade 1 peribronchial inflammation, grade 1.5 perivascular inflammation, and grade 2.5 total lung inflammation.

Close modal
Table IV.

Long-term effects of CpG ODN on peribronchial and perivascular lung inflammationa

Lung Inflammation Scorea
RW/PBSRW/CpG–6wk
Peribronchial 2.67 ± 0.33 1.17 ± 0.17b 
Perivascular 3.17 ± 0.31 1.67 ± 0.25b 
Total 5.83 ± 0.54 2.83 ± 0.38*** 
Lung Inflammation Scorea
RW/PBSRW/CpG–6wk
Peribronchial 2.67 ± 0.33 1.17 ± 0.17b 
Perivascular 3.17 ± 0.31 1.67 ± 0.25b 
Total 5.83 ± 0.54 2.83 ± 0.38*** 
a

Inflammation score was determined by a pathologist blinded to the treatment groups. Peribronchial and perivascular inflammation were graded on a subjective scale of 0, 1, 2, 3, and 4 corresponding to mild, moderate, marked, or severe inflammation respectively with an increment of 0.5 if the inflammation fell between two integers. The total lung inflammation was defined as the sum of peribronchial and perivascular inflammation scores. The values are expressed as mean ± SEM for six animals.

b

, p < 0.01; and ***, p < 0.001 compared with the RW/PBS group.

Airway responsiveness was measured in unrestrained animals using whole body plethysmography (BUXCO, Troy, NY) (23). Mice were placed in the main chamber of the plethysmograph, and baseline readings were taken and averaged for 5 min. Airway reactivity was expressed as fold increase in enhanced pause (PENH) for each concentration of methacholine relative to the PENH values produced by PBS exposure for individual mice. Compared with the RW/PBS group, the RW/CpG-6 wk group demonstrated a 32% reduction in the PENH index at 50 mg/ml dose of methacholine (p < 0.01, Fig. 7).

FIGURE 7.

Prolonged effects of CpG ODN on bronchial hyperresponsiveness. The PENH index was used to measure bronchial hyperresponsiveness in the RW/PBS and RW/CpG-6 wk long term groups. ∗∗, p < 0.01 compared with the RW/PBS group. Values represent the mean ± SEM for six animals.

FIGURE 7.

Prolonged effects of CpG ODN on bronchial hyperresponsiveness. The PENH index was used to measure bronchial hyperresponsiveness in the RW/PBS and RW/CpG-6 wk long term groups. ∗∗, p < 0.01 compared with the RW/PBS group. Values represent the mean ± SEM for six animals.

Close modal

Because administration of CpG ODN dramatically increased the ratio of IFN-γ-IL-4 cells in the lungs, we hypothesized that IFN-γ played a critical role in mediating the effects of CpG ODN in allergic lung inflammation. To test this hypothesis, CpG ODN or PBS was administered 48 h before RW challenge in allergen-sensitized IFN-γ +/+ and IFN-γ −/− BALB/c mice in the short term asthma model. Administration of CpG ODN resulted in a 78% reduction in BAL eosinophil numbers compared with the RW/PBS group in the IFN-γ +/+ mice (p < 0.001, Fig. 8). The degree of inhibition was similar to that observed in the RW/CpG-48 h group (70%, Fig. 1). In contrast, CpG ODN failed to inhibit eosinophil recruitment in IFN-γ −/− mice, indicating that IFN-γ is an essential mediator of the CpG ODN antiinflammatory effect.

FIGURE 8.

Role of IFN-γ in mediating the rapid effects of CpG ODN on eosinophil recruitment. IFN-γ +/+ (WT) or IFN-γ −/− (KO) BALB/c mice (The Jackson Laboratory) were first sensitized with two doses of ragweed and alum on days 0 and 4. CpG ODN or PBS was administered intratracheally 48 h before RW challenge to maximize the inhibitory effects of CpG ODN on allergic lung inflammation. Three days after RW challenge, BALs were performed on each group of mice. Shown are BAL eosinophil cell numbers × 104/ml in the four treatment groups. n = 4 or 5 per group. All values are expressed as mean ± SEM. ∗∗∗, p < 0.001 compared with the corresponding RW/PBS group.

FIGURE 8.

Role of IFN-γ in mediating the rapid effects of CpG ODN on eosinophil recruitment. IFN-γ +/+ (WT) or IFN-γ −/− (KO) BALB/c mice (The Jackson Laboratory) were first sensitized with two doses of ragweed and alum on days 0 and 4. CpG ODN or PBS was administered intratracheally 48 h before RW challenge to maximize the inhibitory effects of CpG ODN on allergic lung inflammation. Three days after RW challenge, BALs were performed on each group of mice. Shown are BAL eosinophil cell numbers × 104/ml in the four treatment groups. n = 4 or 5 per group. All values are expressed as mean ± SEM. ∗∗∗, p < 0.001 compared with the corresponding RW/PBS group.

Close modal

There has been a steady increase in the incidence, morbidity, and mortality caused by allergic asthma. Thus, a form of therapy that could suppress for long periods the lung inflammation found in this disease would be of considerable public health benefit. This study demonstrates for the first time a prolonged effect abrogating allergic lung inflammation in presensitized mice. The intratracheal administration of synthetic oligonucleotides carrying immunostimulatory CpG motifs converted the Th2 cell-mediated allergic response to a dominant Th1 phenotype and significantly reduced eosinophilic lung inflammation, RW-specific IgE production, and bronchial hyperresponsiveness 6 wk after the last dose of CpG ODN.

There are two previous reports (Kline et al. (20); Broide et al. (21)) of CpG ODN having antiallergic properties. Kline et al. administered ODNs along with Schistosoma eggs i.p. in the Th1-prone mouse strain C57BL/6. Six hours after Schistosoma egg Ag challenge, BAL eosinophil were reduced in mice treated with CpG ODN. Broide et al. evaluated the effects of CpG ODN in two models of OVA-sensitized and -challenged BALB/c mice. In the first model, i.p. administration of three 50-μg doses of CpG ODNs 24 h before each of the three OVA challenges inhibited bronchial hyperresponsiveness. In the second model, administration of 100-μg dose(s) of CpG i.p., intranasally, or intratracheally 1 or 6 days before the final OVA challenge inhibited eosinophil recruitment. Thus, both Kline et al. and Broide et al. have demonstrated short term effects of CpG ODN on allergic lung inflammation. As in our study, both groups used nuclease-resistant phosphorothioate ODN, given that their greater half-life is expected to improve activity in vivo. Our study represents an important extension of these reports in that it is the first study demonstrating that CpG ODN reduces immunological and physiological manifestations of allergic asthma for a prolonged period. Our study was conducted in a Th2-prone mouse strain (BALB/c) using an allergen (RW) that is relevant to human allergic asthma. Like Broide et al., we directly delivered CpG ODN to the lungs mimicking nebulizer delivery. Finally, ours is the first study to document that CpG ODNs require IFN-γ to inhibit eosinophilic lung inflammation.

Our experiments indicate that CpG ODN suppress allergic lung inflammation optimally if delivered 2 days before allergen challenge. Indeed, no benefit was observed when the ODN were coadministered with the allergen. These findings are consistent with other evidence that CpG ODN require 2–3 days to induce an optimal Th1-mediated immune response in vivo and support the hypothesis that CpG ODN trigger an immunomodulatory cascade that matures over a period of several days (25). Of interest, once CpG ODN established a RW-specific Th1 bias in the lungs, the preferential induction of a Th1 response persisted for at least 6 wk. This long term preferential induction of Th1 response was associated with a reduction in the number of cells secreting IL-4, suggesting inhibition of Th2 response. In keeping with the known opposing effects of Th1 and Th2 cytokines on isotype switching by B cells, the reduction in IL-4 and increase in IFN-γ was associated with a reduction in IgE- and IgG1-secreting cells and an increase in the number of IgG2a-secreting cells in the spleen and lungs. These effects on Ig production may have relevance to the prolonged inhibitory effects of CpG ODN on allergic lung inflammation because prior studies indicate that allergen-specific IgE and IgG1, but not IgG2a, augments allergic lung inflammation (10, 26). The long term effect on the IFN-γ-IL-4 ratio, combined with the increased Th1 memory response to allergen and the absence of an effect in IFN-γ −/− mice, indicates that the stimulation of Th1 cells producing IFN-γ plays a key role in maintaining the antiinflammatory effects of CpG ODN in the lung.

In the present study, CpG ODN increased the number of IFN-γ-producing cells in the lungs 6 wk after the last dose of CpG ODN. This could reflect the generation of resident memory cells in the lungs or the recruitment of Th1 cells from a systemic reservoir to the lung following each intratracheal RW challenge. It is likely that after allergen challenge, Th1 memory cells are recruited in greater numbers to the lungs from systemic “reservoir” organs such as spleen. We and others have shown that the CC chemokines, RANTES and macrophage-inflammatory protein 1α, are produced in asthma and allergic inflammation (27, 28, 29). These CC chemokines are also efficient chemoattractants for Th1 cells and have been shown to induce a dose-dependent transmigration of Th1 but not of Th2 cells (30, 31). Thus, in the long term asthma model in our study, the final intratracheal RW allergen challenge may have initiated the recruitment of Th1 cells to the lung by increasing the intrapulmonary levels of RANTES and macrophage-inflammatory protein 1-α.

The ability of CpG ODN to stimulate IL-12 production may be a key factor mediating the prolonged effects of CpG ODN. We and others have recently shown that IL-12, a cytokine that promotes Th1 differentiation and production of IFN-γ, inhibits eosinophil recruitment, decreases IgE levels, and suppresses BHR in murine models of allergic asthma when it is given systemically within 4–72 h of allergen challenge (17, 32, 33, 34, 35). More recently, we have found that intratracheal administration of IL-12 with ragweed in the mouse model of asthma has long term effects that inhibit eosinophil recruitment (36). Prior studies indicate that systemic administration of IL-12 at the time of live parasite egg inoculation or at the time of parasite Ag injections leads to long term vaccine adjuvant effects that decrease footpad swelling and pathology induced by a parasite challenge (37, 38, 39). Some of these studies reported persistence of Ag-specific Th1 memory a few weeks after immunization with Ag and IL-12 (37, 38, 39). In one of these long term studies, Ag-specific Th1 memory persisted for 8 wk (38). Because our study indicates that CpG ODNs stimulate long term Ag-specific Th1 memory, it is possible that this is mediated by induction of IL-12 production (20).

In the current study, CpG ODN was shown to augment IFN-γ production and, in the long term protocol, reduce the number of cells producing IL-4. In mouse models of asthma, individual treatment with anti-IL-4 and intratracheal administration of IFN-γ and IL-12 have been shown to inhibit allergic lung inflammation (14, 33, 40). Treatment with exogenous Th1-promoting or Th2-limiting agents, however, may not be sufficiently efficacious in the treatment of human asthma or may pose toxic consequences. Even though animal studies of IFN-γ have been encouraging, clinical trials of IFN-γ in patients with allergic rhinitis and asthma have not yielded promising results (41, 42, 43). Use of exogenous IFN-γ may also be limited by its side effects, which include influenza-like symptoms including fever and fatigue (43). Systemically administered IL-12 has also been reported to produce toxicity (44), although these effects may be avoidable by the use of low dose IL-12 administered intratracheally or via other nonsystemic routes of administration. The ability of CpG ODN to stimulate the combination of Th1-promoting and Th2-limiting effects suggests a potent therapeutic potential in the treatment of allergic diseases. Also, because endogenously produced cytokines are likely to be homeostatically regulated, CpG ODN may produce less toxicity than administration of exogenous cytokines. However, because of the limited data from clinical trials and the potential differences between effects in animal studies and in patients with asthma, the efficacy of anti-IL-4, IL-12, and CpG ODN as therapeutic agents in asthma remains to be determined.

The ability of intratracheally administered CpG ODN to provide long term protection against allergic lung inflammation and alter the balance between IL-4 and IFN-γ suggest that intratracheally administered CpG ODN may have therapeutic benefit in asthma. Many current asthma therapies utilize inhalation administration of medications to maximize patient compliance and minimize systemic toxicity. Pulmonary administration of CpG ODN may provide more effective inhibition of allergic lung inflammation than systemic administration. In a recent study by Erb et al. (45), intranasal infection with bacillus Calmette-Guérin (BCG) produced significantly greater inhibition of allergic airway eosinophilia than i.p. or s.c. infection. Studies in our laboratory indicate that intratracheal administration of recombinant IL-12 is 100-fold more effective in suppressing allergic lung inflammation than the same dose of IL-12 delivered systemically.4 If CpG ODN is also more efficacious when administered intratracheally, then CpG ODN delivery by this route might minimize the dose of CpG ODN required for treatment, thereby reducing potential side effects. Moreover, since delivery of CpG ODN results in long term reduction in allergic asthma, this method of administration might require infrequent dosing. With regard to potential toxicity, we found that 35 μg of CpG ODN administered intratracheally to naive BALB/c mice did not induce pulmonary inflammation as determined in BAL fluids 4 and 48 h later. This is in contrast to the results of Schwartz et al., who described a neutrophilic inflammation in C3H/BfeJ mice 4 h after intratracheal administration of CpG ODN (46). Whether this difference is strain specific or correlates with differences in sequence of the CpG ODN remains to be determined.

Purified bacterial DNA has effects similar to CpG ODN on the immune system. Due to differences in the frequency of CpG use and methylation, CpG motifs are 20-fold more common in the genomes of bacteria than vertebrates (47). Evidence suggests that the mammalian immune system recognizes these motifs, rapidly stimulating the host to mount a Th1-dominated innate immune response (19). In this context, our findings support the hypothesis of Shirakawa et al. (48) that bacterial lung infections may protect against the development of asthma. They report that the rising incidence of asthma in industrialized countries is paralleled by a decline in the incidence of childhood mycobacterial infections and a reduction in the ability of children to respond to tuberculin testing. Erb et al. (45) reported that in a mouse model, active BCG pulmonary infection inhibited allergic lung inflammation. There are at least two important differences between their study and our study (45). First, BCG infection was initiated before allergic sensitization, unlike CpG ODN treatment after sensitization in our study (45). Thus, the goal of the study of Erb et al. was to determine whether BCG infection could prevent the development of asthma in normal children. In contrast, our study was designed to determine whether CpG ODN could deviate the immune response from a Th2 bias in individuals who are already sensitized to allergens to a protective Th1 response. Second, BCG infection of the lung did not decrease production of allergen-specific IgE or IgG1, both of which are desirable effects for treating asthma (10, 26, 49, 50). Because bacterial DNA is enriched in CpG motifs, release of bacterial DNA during infection might act through a CpG ODN-mediated mechanism to suppress allergic inflammation. Definitive conclusions on the role of pulmonary bacterial infection in allergic inflammation, the release of biologically active DNA by degrading bacteria in the lungs, and conflicting epidemiological studies await clarification (51).

In summary, this is the first report of an agent capable of inducing a prolonged inhibition of allergic lung inflammation in a presensitized mouse model of asthma. Intratracheally administered CpG ODN preferentially stimulated the production of Th1 cytokines and suppressed eosinophilic airway inflammation, allergen-specific IgE production, and bronchial hyperresponsiveness for a prolonged period. Thus, local administration of CpG ODN deserves further study as a potential treatment for asthma.

We thank Joani Zary, East Carolina University School of Medicine, Greenville, NC.

1

This work was supported by the Department of Internal Medicine, University of Texas Medical Branch, and the James W. McLaughlin Fellowship Fund.

3

Abbreviations used in this paper: RW, ragweed allergen; ODN, oligodeoxynucleotides; BAL, bronchoalveolar lavage; ELISPOT, enzyme-linked immunospot; PENH, enhanced pause; BCG, bacillus Calmette-Guérin.

4

S. Sur et al. Submitted for publication.

1
Sears, M. R..
1991
. Worldwide trends in asthma mortality.
Bull. Int. Union Tuberc.
66
:
79
2
MMWR. 1996. Asthma mortality and hospitalizations among children and young adults: United States. MMWR 45:350.
3
Martin, L. B., H. Kita, K. M. Leiferman, G. J. Gleich.
1996
. Eosinophils in allergy: role in disease, degranulation, and cytokines.
Int. Arch. Allergy Immunol.
109
:
207
4
Sur, S., C. Adolphson, G. J. Gleich.
1993
. Eosinophils: biochemical and cellular aspects. E. Middleton, Jr, and C. E. Reed, Jr, and E. F. Ellis, Jr, and N. F. Adkinson, Jr, and J. W. Yunginger, Jr, and W. W. Busse, Jr, eds. In
Allergy: Principles and Practice
Vol. 1
:
169
Mosby, St. Louis.
5
Gleich, G. J., D. B. Jacoby, A. D. Fryer.
1995
. Eosinophil-associated inflammation in bronchial asthma: a connection to the nervous system.
Int. Arch. Allergy Immunol.
107
:
205
6
Gundel, R. H., L. G. Letts, G. J. Gleich.
1991
. Human eosinophil major basic protein induces airway constriction and airway hyperresponsiveness in primates.
J. Clin. Invest.
87
:
1470
7
White, S. R., S. Ohno, N. M. Munoz, G. J. Gleich, C. Abrahams, J. Solway, A. R. Leff.
1990
. Epithelium-dependent contraction of airway smooth muscle caused by eosinophil MBP.
Am. J. Physiol.
259
:
L294
8
Gleich, G. J., G. L. Jacob.
1975
. Immunoglobulin E antibodies to pollen allergens account for high percentages of total immunoglobulin E protein.
Science
190
:
1106
9
Solley, G. O., G. J. Gleich, R. E. Jordon, A. L. Schroeter.
1976
. The late phase of the immediate wheal and flare skin reaction: Its dependence upon IgE antibodies.
J. Clin. Invest.
58
:
408
10
Coyle, A. J., K. Wagner, C. Bertrand, S. Tsuyuki, J. Bews, C. Heusser.
1996
. Central role of immunoglobulin (Ig) E in the induction of lung eosinophil infiltration and T helper 2 cell cytokine production: inhibition by a non-anaphylactogenic anti-IgE antibody.
J. Exp. Med.
183
:
1303
11
Ohnishi, T., S. Sur, D. S. Collins, J. E. Fish, G. J. Gleich, S. P. Peters.
1993
. Eosinophil survival activity identified as interleukin-5 is associated with eosinophil recruitment and degranulation and lung injury twenty-four hours after segmental antigen lung challenge.
J. Allergy Clin. Immunol.
92
:
607
12
Nakajima, H., I. Iwamoto, S. Tomoe, R. Matsumura, H. Tomioka, K. Takatsu, S. Yoshida.
1992
. CD4+ T-lymphocytes and interleukin-5 mediate antigen-induced eosinophil infiltration into the mouse trachea.
Am. Rev. Respir. Dis.
146
:
374
13
Robinson, D. S., Q. Hamid, S. Ying, A. Tsicopoulos, J. Barkans, A. M. Bentley, C. Corrigan, S. R. Durham, A. B. Kay.
1992
. Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma.
N. Engl. J. Med.
326
:
298
14
Coyle, A. J., G. Le Gros, C. Bertrand, S. Tsuyuki, C. H. Heusser, M. Kopf, G. P. Anderson.
1995
. Interleukin-4 is required for the induction of lung Th2 mucosal immunity.
Am. J. Respir. Cell Mol. Biol.
13
:
54
15
Brusselle, G., J. Kips, G. Joos, H. Bluethmann, R. Pauwels.
1995
. Allergen-induced airway inflammation and bronchial responsiveness in wild-type and interleukin-4-deficient mice.
Am. J. Respir. Cell Mol. Biol.
12
:
254
16
Iwamoto, I., H. Nakajima, H. Endo, S. Yoshida.
1993
. Interferon γ regulates antigen-induced eosinophil recruitment into the mouse airways by inhibiting the infiltration of CD4+ T cells.
J. Exp. Med.
177
:
573
17
Sur, S., J. Lam, P. Bouchard, A. Sigounas, D. Holbert, W. J. Metzger.
1996
. Immunomodulatory effects of IL-12 on allergic lung inflammation depend on timing of doses.
J. Immunol.
157
:
4173
18
Krieg, A. M., A. K. Yi, S. Matson, T.J. Waldschmidt, G. A. Bishop, R. Teasdale, G. A. Koretzky, D. M. Klinman.
1995
. CpG motifs in bacterial DNA trigger direct B-cell activation.
Nature
374
:
546
19
Klinman, D. M., A. K. Yi, S. L. Beaucage, J. Conover, A. M. Krieg.
1996
. CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma.
Proc. Natl. Acad. Sci. USA
93
:
2879
20
Kline, J. N., T. J. Waldschmidt, T. R. Businga, J. E. Lemish, J. V. Weinstock, P. S. Thorne, A. M. Krieg.
1998
. Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma.
J. Immunol.
160
:
2555
21
Broide, D., J. Schwarze, H. Tighe, T. Gifford, M. D. Nguyen, S. Malek, J. Van Uden, E. Martin-Orozco, E. W. Gelfand, E. Raz.
1998
. Immunostimulatory DNA sequences inhibit IL-5, eosinophilic inflammation, and airway hyperresponsiveness in mice.
J. Immunol.
161
:
7054
22
Hunt, L. W., G. J. Gleich, T. Ohnishi, D. A. Weiler, E. S. Mansfield, H. Kita, S. Sur.
1994
. Endotoxin contamination causes neutrophilia following pulmonary allergen challenge.
Am. J. Respir. Crit. Care Med.
149
:
1471
23
Hamelmann, E., J. Schwarze, K. Takeda, A. Oshiba, G. L. Larsen, C. G. Irvin, E. W. Gelfand.
1997
. Noninvasive measurement of airway responsiveness in allergic mice using barometric plethysmography.
Am. J. Respir. Crit. Care Med.
156
:
766
24
Ashcroft, T., J. M. Simpson, V. Timbrell.
1988
. Simple method of estimating severity of pulmonary fibrosis on a numerical scale.
J. Clin. Pathol.
41
:
467
25
Krieg, A. M., L. Love-Homan, J. T. Harty.
1998
. CpG DNA induces sustained IL-12 expression in vivo and resistance to Listeria monocytogenes challenge.
J. Immunol.
161
:
2428
26
Oshiba, A., E. Hamelmann, K. Takeda, K. L. Bradley, J. E. Loader, G. L. Larsen, E. W. Gelfand.
1996
. Passive transfer of immediate hypersensitivity and airway hyperresponsiveness by allergen-specific immunoglobulin (Ig) E and IgG1 in mice.
J. Clin. Invest.
97
:
1398
27
Alam, R., J. York, M. Boyars, S. Stafford, J. A. Grant, J. Lee, P. Forsythe, T. Sim, N. Ida.
1996
. Increased MCP-1, RANTES, and MIP-1α in bronchoalveolar lavage fluid of allergic asthmatic patients.
Am. J. Respir. Crit. Care Med.
153
:
1398
28
Sur, S., H. Kita, G. J. Gleich, T. C. Chenier, L. W. Hunt.
1996
. Eosinophil recruitment is associated with IL-5, but not with RANTES, twenty-four hours after allergen challenge.
J. Allergy Clin. Immunol.
97
:
1272
29
Teran, L. M., N. Noso, M. Carroll, D. E. Davies, S. Holgate, J. M. Schroder.
1996
. Eosinophil recruitment following allergen challenge is associated with the release of the chemokine RANTES into asthmatic airways.
J. Immunol.
157
:
1806
30
Siveke, J. T., A. Hamann.
1998
. Cutting edge: T helper 1 and T helper 2 cells respond differentially to chemokines.
J. Immunol.
160
:
550
31
Schrum, S., P. Probst, B. Fleischer, P. F. Zipfel.
1996
. Synthesis of the CC-chemokines MIP-1-α, MIP-1-β, and RANTES is associated with a type 1 immune response.
J. Immunol.
157
:
3598
32
Gavett, S. H., D. J. O’Hearn, X. Li, S. K. Huang, F. D. Finkelman, M. Wills-Karp.
1995
. Interleukin 12 inhibits antigen-induced airway hyperresponsiveness, inflammation, and Th2 cytokine expression in mice.
J. Exp. Med.
182
:
1527
33
Schwarze, J., E. Hamelmann, G. Cieslewicz, A. Tomkinson, A. Joetham, K. Bradley, E. W. Gelfand.
1998
. Local treatment with IL-12 is an effective inhibitor of airway hyperresponsiveness and lung eosinophilia after airway challenge in sensitized mice.
J. Allergy Clin. Immunol.
102
:
86
34
Kips, J.C., G. J. Brusselle, G. F. Joos, R. A. Peleman, J. H. Tavernier, R. R. Devos, R. A. Pauwels.
1996
. Interleukin-12 inhibits antigen-induced airway hyperresponsiveness in mice.
Am. J. Respir. Crit. Care Med.
153
:
535
35
Iwamoto, I., K. Kumano, M. Kasai, K. Kurasawa, A. Nakao.
1996
. Interleukin-12 prevents antigen-induced eosinophil recruitment into mouse airways.
Am. J. Respir. Crit. Care Med.
154
:
1257
36
Sur, S., S. Siddique, R. Alam, W. J. Mileski, P. Bouchard, G. Sarkar, A. Sigounas, J. S. Wild.
1999
. Strategies for the development of vaccines for asthma.
J. Allergy Clin. Immunol.
103
:
S107
(Abstr.).
37
Afonso, L. C., T. M. Scharton, L. Q. Vieira, M. Wysocka, G. Trinchieri, P. Scott.
1994
. The adjuvant effect of interleukin-12 in a vaccine against Leishmania major.
Science
263
:
235
38
Wynn, T. A., A. W. Cheever, D. Jankovic, R. W. Poindexter, P. Caspar, F. A. Lewis, A. Sher.
1995
. An IL-12-based vaccination method for preventing fibrosis induced by schistosome infection.
Nature
376
:
594
39
Wynn, T. A., A. Reynolds, S. James, A. W. Cheever, P. Caspar, S. Hieny, D. Jankovic, M. Strand, A. Sher.
1996
. IL-12 enhances vaccine-induced immunity to schistosomes by augmenting both humoral and cell-mediated immune responses against the parasite.
J. Immunol.
157
:
4068
40
Nakajima, H., I. Iwamoto, S. Yoshida.
1993
. Aerosolized recombinant interferon-γ prevents antigen-induced eosinophil recruitment in mouse trachea.
Am. Rev. Respir. Dis.
148
:
1102
41
Boguniewicz, M., R. J. Martin, D. Martin, U. Gibson, A. Celniker, M. Williams, D. Y. Leung.
1995
. The effects of nebulized recombinant interferon-γ in asthmatic airways.
J. Allergy Clin. Immunol.
95
:
133
42
Scholz, D., K. Brandt-Hofflin, H. L. Hahn.
1990
. [Effect of recombinant gamma interferon on allergic skin reaction and bronchoconstriction] [in German].
Pneumologie
44
: (Suppl. 1):
431
43
Li, J. T., J. W. Yunginger, C. E. Reed, H. S. Jaffe, D. R. Nelson, G. J. Gleich.
1990
. Lack of suppression of IgE production by recombinant interferon γ: a controlled trial in patients with allergic rhinitis.
J. Allergy Clin. Immunol.
85
:
934
44
Leonard, J. P., M. L. Sherman, G. L. Fisher, L. J. Buchanan, G. Larsen, M. B. Atkins, J. A. Sosman, J. P. Dutcher, N. J. Vogelzang, J. L. Ryan.
1997
. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-γ production.
Blood
90
:
2541
45
Erb, K. J., J. W. Holloway, A. Sobeck, H. Moll, G. Legros.
1998
. Infection of mice with Mycobacterium bovis-bacillus Calmette-Guérin (BCG) suppresses allergen-induced airway eosinophilia.
J. Exp. Med.
187
:
561
46
Schwartz, D. A., T. J. Quinn, P. S. Thorne, S. Sayeed, A. K. Yi, A. M. Krieg.
1997
. CpG motifs in bacterial DNA cause inflammation in the lower respiratory tract.
J. Clin. Invest.
100
:
68
47
Cardon, L. R., C. Burge, D. A. Clayton, S. Karlin.
1994
. Pervasive CpG suppression in animal mitochondrial genomes.
Proc. Natl. Acad. Sci. USA
91
:
3799
48
Shirakawa, T., T. Enomoto, S. Shimazu, J. M. Hopkin.
1997
. The inverse association between tuberculin responses and atopic disorder [see comments].
Science
275
:
77
49
Boulet, L. P., K. R. Chapman, J. Cote, S. Kalra, R. Bhagat, V. A. Swystun, M. Laviolette, L. D. Cleland, F. Deschesnes, J. Q. Su, A. DeVault, R. B. Fick, Jr, D. W. Cockcroft.
1997
. Inhibitory effects of an anti-IgE antibody E25 on allergen-induced early asthmatic response [see comments].
Am. J. Respir. Crit. Care Med.
155
:
1835
50
Fahy, J. V., H. E. Fleming, H. H. Wong, J. T. Liu, J. Q. Su, J. Reimann, R. B. Fick, Jr, H. A. Boushey.
1997
. The effect of an anti-IgE monoclonal antibody on the early- and late-phase responses to allergen inhalation in asthmatic subjects [see comments].
Am. J. Respir. Crit. Care Med.
155
:
1828
51
Strannegard, I. L., L. O. Larsson, G. Wennergren, O. Strannegard.
1998
. Prevalence of allergy in children in relation to prior BCG vaccination and infection with atypical mycobacteria.
Allergy
53
:
249