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Division of Allergy and Immunology, Department of Internal Medicine, University of Texas Medical Branch, Galveston, TX 77555; and
Retroviral Immunology Section, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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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- -/- mice failed to
inhibit eosinophil recruitment, indicating a critical role of IFN-
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. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- 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- 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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice, 6–8 wk old, were purchased from the Harlan
Laboratories (Indianapolis, IN) to perform all experiments except those
requiring IFN- -/- and IFN- +/+ mice. The latter (IFN- -/-
and IFN- +/+ 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.
Oligonucleotides
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.
Ragweed
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.
Experimental design
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.
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Evaluation of the role of IFN- in mediating the rapid effects of
CpG ODN
IFN- +/+ and IFN- -/- 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.
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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).
Splenocyte recall response
Splenocytes were incubated at 5 x 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- levels with a two-site immunoenzymetric assay using
anti-IFN- Abs (clones R4-6A2 and XMG1.2, PharMingen, San
Diego, CA).
Cytokine ELISPOT assays
Immulon 2 microtiter plates (96-well) were coated with 10
µg/ml anti-IFN- (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 x 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- (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.
Ragweed-specific ELISA and ELlSPOT assays
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.
Fold increase in the ratio of IFN--IL-4-secreting cells
The ELISPOT data were used to calculate the ratio of
IFN--IL-4-secreting cells. The fold increases in the ratio of
IFN- 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--IL-4 ratios were calculated separately.
Pulmonary function testing
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).
Lung histology
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.
Data analysis
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1A,
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. 1B, 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).
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) 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.
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-IL-4
Cytokine ELISPOT assays were used to monitor the number of cells
actively secreting IFN- 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. 2A). When
administered 48 h before allergen challenge, CpG ODN significantly
increased the number of IFN- producing cells in the spleen
(p < 0.05; Fig. 2A). This altered
the cytokine milieu by increasing the ratio of IFN--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--producing cells in the lungs (Fig. 2B) and increased
the ratio of IFN--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.
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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--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--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.
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C, splenocytes derived from both the RW/CpG-2 days and
RW/CpG-6 wk groups responded vigorously to the recall Ag with IFN-
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-. Thus, in
addition to increasing the ratio of IFN--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. 4A). 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--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.
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to induce isotype switching from IgG1 to IgG2a, CpG ODN
decreased the number of IgG1-producing cells (Fig. 4C) and
increased the number of IgG2a-secreting cells in the lungs (Fig. 4D). 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--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. CpG ODN has long term antiallergic effects
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.
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A (naive), 6B (RW/PBS) and 6C
(RW/CpG-6 wk) show representative hematoxylin and eosin-stained
peribronchial and perivascular areas of the lungs (x40). 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).
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Because administration of CpG ODN dramatically increased the ratio
of IFN--IL-4 cells in the lungs, we hypothesized that IFN- 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- +/+ and
IFN- -/- 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- +/+
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- -/- mice,
indicating that IFN- is an essential mediator of the CpG ODN
antiinflammatory effect.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- 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- 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--IL-4 ratio, combined with the increased Th1 memory response to
allergen and the absence of an effect in IFN- -/- mice, indicates
that the stimulation of Th1 cells producing IFN- 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--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 1
, 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-.
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-, 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- 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- 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- have
been encouraging, clinical trials of IFN- in patients with allergic
rhinitis and asthma have not yielded promising results (41, 42, 43). Use of
exogenous IFN- 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- 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.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sanjiv Sur, Department of Internal Medicine, Division of Allergy and Immunology, Route 0762, 409 CSB, 301 University Blvd., University of Texas Medical Branch, Galveston, TX 77555-0762. E-mail address:
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.
Received for publication December 8, 1998. Accepted for publication March 1, 1998.
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