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Department of Pharmacology and Pathophysiology, Utrecht University, Utrecht, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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- and IL-2-producing Th1 cells or to
the IL-4- and IL-5-producing Th2 cells (2). APCs appear to be important
in the skewing of Th cell differentiation. First, although still
controversial, costimulatory molecules on the APCs are suggested to be
associated with the development of Th1 (B7-1/CD80) or Th2 (B7-2/CD86)
cells (3, 4). Second, soluble factors such as cytokines derived from
APCs are thought to play a role in the Th subset development. IL-12
promotes Th1 differentiation, enhances production of Th1 cytokines such
as IL-2 and IFN- (5, 6, 7), and inhibits Th2 cytokine synthesis
(8, 9, 10). IL-4 is essential for the development of Th2 cells and
inhibits the proliferation of Th1 cells (2). Recently, a new cytokine
IL-18, also called IFN--inducing factor, has been cloned (11). IL-18
can be synthesized by Küpffer cells and activated macrophages
(11), and is of particular interest, since it potentiates the
IL-12-driven Th1 development in BALB/c mice (12), and in combination
with IL-12 strongly induces IFN- production (11, 12, 13, 14). These studies
were performed in vitro, and not much is known about the effectiveness
in vivo on the differentiation into either Th1 or Th2 cells. In mouse models of Ag-induced airway inflammation, IL-4 and IL-5 have been shown to play a crucial role in the development of IgE, airway hyperresponsiveness, and eosinophilic infiltration (15, 16, 17, 18), indicating a regulatory role for Th2-like cells. In previous studies, using a mouse model of allergic asthma, we have shown that after sensitization, Ag-specific T cells are present in the lung and lung-draining lymph nodes and display a typical Th0-like cytokine production pattern upon restimulation in vitro (19). The induction of airway symptoms by repeated Ag inhalation was associated with the appearance of Ag-specific Th2-like cells in lung and lymph nodes.3 This model can be used to investigate the in vivo differentiation of T cells into Th2-like cells upon Ag inhalation and relate this to disease symptoms.
Currently, we investigated whether IL-12 and IL-18 could interfere in the development of Th2-like cells and the implications for airway symptoms. Therefore, sensitized mice were treated during the challenge period with IL-12, IL-18, or a combination of IL-12 and IL-18. The cytokine production patterns were determined from Ag-stimulated T cells in the lymph nodes and lung tissue. In addition, these results were compared with allergic airway symptoms such as IgE, airway hyperresponsiveness, and cellular infiltration in the lungs.
The present study shows that only treatment with a combination of IL-12 and IL-18 could inhibit the development of Th2-like cells, which was associated with decreased IgE levels and inhibition of airway hyperresponsiveness and cellular inflammation. This effect was not observed after treatment with either IL-12 or IL-18, indicating a synergistic effect of IL-12 and IL-18.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animal care and use were performed in accordance with the guidelines of Dutch Committee of Animal Experiments. Specific pathogen-free male BALB/c mice (6 wk) were obtained from the breeding colony of Central Animal Laboratory (Utrecht, The Netherlands). The mice were housed in macrolon cages and provided with food and water ad libitum.
Immunization protocol
Active sensitization was performed without an adjuvant by giving seven i.p. injections of 10 µg OVA (grade V) in 0.5 ml pyrogen-free saline on alternate days (one injection per day). Three weeks after the last injection, the mice were exposed to eight OVA (2 mg/ml) or eight saline aerosol challenges for 5 min on consecutive days (one aerosol per day). The aerosol was performed in a plexiglas exposure chamber (5 L) coupled to a Jet nebulizer (Pari IS-2 Jet nebulizer; PARI Respiratory Equipment, Richmond, VA; particle size 2–3 µm) driven by compressed air at a flow rate of 6 L/min. Aerosol was given in groups composed maximally of six animals.
Treatment with IL-12 and/or IL-18 in vivo
OVA-sensitized BALB/c mice were divided in four groups of 16 mice; 8 mice per group received saline challenge and 8 mice per group received OVA challenge. Aerosol was given as described above. One group of mice received i.p. 0.25 ml saline (vehicle group); a second group received i.p. murine rIL-12 (100 ng in 0.25 ml saline); a third group received i.p. murine rIL-18 (500 ng in 0.25 ml saline); and a fourth group received i.p. a combination of murine rIL-12 and murine rIL-18 (respectively, 100 ng and 500 ng in 0.25 ml saline). Treatment started 30 min before the first aerosol and was given once per day before the OVA or saline challenge. Murine rIL-12 and murine rIL-18 were purchased by Sanvertech (Heerhugowaard, The Netherlands). The endotoxin levels were less than 0.1 ng/µg IL-12 or IL-18. Based on a study by Yoshimoto and colleagues (14), we chose the concentrations of IL-12 and IL-18.
In each mice, airway responsiveness to methacholine, IgE levels in serum, cellular infiltration in the bronchoalveolar lavage, and T cell responses in lung-draining lymph nodes and lung tissue were measured 24 h after the last challenge.
Airway hyperresponsiveness
Using barometric whole-body plethysmography (Buxco, Sharon, CT), responses to inhaled methacholine in conscious, unrestrained mice were measured as described previously (20). As an index of airway responsiveness, increases in enhanced pause (Penh) were measured. In short, mice were placed in a whole-body chamber, and basal readings were obtained and averaged for 3 min. Aerosolized saline, followed by increasing concentrations of methacholine (ranging from 1.6–50 mg/ml), was nebulized for 3 min, and readings were taken and averaged for 3 min after each nebulization. Airway responsiveness was expressed as the Penh per dose methacholine.
Serum levels of IgE
Directly after the dose-response curve with methacholine, the mice were injected with an overdose of pentobarbitone (0.5 g/kg body weight). Blood samples were obtained from the mice via a cardiac puncture and centrifuged for 10 min at 14,000 rpm. Serum was collected and samples were kept at -20°C until IgE levels were measured.
Briefly, microplates (96 wells; Nunc A/S, Roskilde, Denmark) were coated with rat anti-mouse IgE (2 µg/ml PBS) at 4°C for 24 h. The ELISA was performed at room temperature. After blocking with ELISA buffer (containing 50 mM Tris, 2 mM EDTA, 136.9 mM NaCl, 0.05% Tween-20, and 0.5% BSA, pH 7.2) for 1 h, appropriate dilutions of the samples and standard, diluted in ELISA buffer, were added for 2 h. After incubation, 1 µg/ml biotinylated anti-mouse IgE was added for 1.5 h, followed by incubation with 0.33 µg/ml peroxidase-conjugated streptavidin for 1 h. The substrate o-phenylenediamine-dichloride (0.4 mg/ml) in PBS containing 0.012% hydrogen peroxide was added. After approximately 15 min, the reaction was stopped by adding 4 M H2SO4. Subsequently, OD was measured at 492 nm, using a Titertek Multiskan.
Ab titers of samples were calculated by comparison with an internal total IgE reference serum standard that was serially diluted. Detection limit of the ELISA was 0.78 ng/ml IgE.
Bronchoalveolar lavage
Bronchoalveolar lavage was performed as described previously (17). The airways of the mice were cannulated and 5 times lavaged through this cannula with 1-ml aliquots of pyrogen-free saline warmed to 37°C. The total numbers of lavage cells were determined, and cells in cytospin preparations were differentiated (Diff-Quick; Merz and Dade, Düdingen, Switzerland) into mononuclear cells, eosinophils, and neutrophils by standard morphology. Per cytospin preparation, at least 200 cells were counted and the absolute number of each cell type was calculated. To evaluate differences between OVA-challenged mice and saline-challenged mice for the different treatments, the numbers of the various cell types were tested with an analysis of variance. For the very low number of eosinophils and neutrophils in saline-challenged mice, a Poisson distribution was assumed, and for differences between treatment groups, Fisher’s exact test was used.
Stimulation of thoracic lymph nodes and lung cells in vitro
Cytokine production by Ag-stimulated T cells derived from both thoracic lymph nodes and lung tissue was determined, as described previously (19). Mice were injected with an overdose of pentobarbitone (0.5 g/kg body weight). The lungs were lavaged (as described below) and perfused via the right ventricle with 4 ml saline containing 100 U/ml heparin to remove any blood and intravascular leukocytes. The thoracic lymph nodes, derived from the paratracheal and parabronchial regions, and the lungs were removed. The lymph nodes were transferred to cold PBS and gently homogenized on a 70-µm cell strainer (Falcon, Lelystad, The Netherlands) to obtain a single cell suspension. The lungs were minced and digested with 3 ml RPMI containing 2.4 mg/ml collagenase, 1 mg/ml DNase, and 50 µg/ml gentamicin for 30 min at 37°C. The cell suspension was resuspended and filtered through a 70-µm cell strainer with 10 ml RPMI containing 20% heat-inactivated FCS. The lymph node and lung cell suspensions were washed and resuspended in culture medium (RPMI 1640 containing 10% FCS, 1% glutamax I, 50 µg/ml gentamicin, and 50 mM ß-mercaptoethanol), and total cell number was counted. Cells (viability >95%, 2 x 105 lymph node cells/well and 8 x 105 lung cells/well) were plated in round-bottom 96-well plates (Costar, Badhoevedorp, The Netherlands) in a volume of 200 µl. The cells were cultured for 5 days with different stimuli: in the presence of OVA (10 µg/ml), or medium only. The cells were cultured at 37°C with 5% CO2 in humidified air. Each in vitro stimulation was performed in triplicate. Supernatants were harvested, pooled per stimulation, and kept at -20°C until cytokine levels were determined by ELISA.
The IFN-, IL-4, and IL-5 ELISAs were performed according to the
instructions of the manufacturer (PharMingen, San Diego, CA). The
detection limits of the ELISAs were 156 pg/ml for IFN-, 15.6 pg/ml
for IL-4, and 31.3 pg/ml for IL-5.
Stimulation of thoracic lymph nodes and lung cells in the presence of IL-12 and/or IL-18
In a different set of experiments, thoracic lymph node and lung cells were isolated from OVA-sensitized and OVA-challenged mice. The cells from different mice (3, 4, 5, 6) were pooled and cultured, as described above, in the presence (5 ng/ml) or absence of IL-12, IL-18, or a combination of IL-12 and IL-18.
Data analysis
Unless stated otherwise, data are expressed as mean ± SEM and evaluated using an analysis of variance, followed by a post hoc comparison between groups. A probability value of p < 0.05 was considered statistically significant. Statistical analyses were conducted using Systat, version 5.03 (NaG, Oxford, U.K.).
Chemicals
OVA (chicken egg albumin, crude grade V) and o-phenylenediamine-dihydrochloride were purchased from Sigma (St. Louis, MO); PBS, RPMI, FCS, gentamicin, and glutamax I were purchased from Life Technologies (Merelbeke, Belgium); pentobarbitone (nembutal) was purchased from Sanofi Sante B.V. (Maassluis, The Netherlands); heparin was purchased from Leo Pharmaceuticals (Weesp, The Netherlands); collagenase and DNase were purchased from Boehringer Mannheim (Mannheim, Germany); saline was from B. Braun Medical B.V. (Oss, The Netherlands); and methacholine (acetyl-ß-methylcholine) was purchased from Janssen Chimica (Beerse, Belgium). Mouse IgE reference serum was purchased from ICN Biomedicals (Aurora, OH); anti-mouse IgE and biotinylated anti-mouse IgE were purchased from PharMingen (San Diego, CA); and Diff-Quick was purchased from Merz and Dade.
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Results |
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IgE levels were determined in serum of all mice. No significant differences in IgE levels were observed between the groups of saline-challenged mice.
In the OVA-challenged vehicle-treated mice, the levels of IgE were
significantly increased (388%, p < 0.01) compared
with saline-challenged mice (Fig. 1
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OVA-challenged mice treated with IL-12 or IL-18 also showed a
significant increase (respectively 332 and 268%, p <
0.01) in IgE levels compared with the saline-challenged controls.
Treatment with a combination of IL-12 and IL-18 in OVA-challenged mice
significantly (p < 0.01) inhibited the IgE
up-regulation back to the levels observed in saline-challenged mice.
Therefore, the combination of IL-12 and IL-18 was able to inhibit the
up-regulated IgE levels.
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Airway responsiveness in vivo to aerosolized methacholine was
measured 24 h after the last challenge in conscious, unrestrained
mice. For all mice, a complete dose-response curve to methacholine
ranging from 1.6 to 50 mg/ml methacholine was constructed. In all
groups of mice, no significant differences were observed in the basal
Penh and saline aerosol-induced Penh values between OVA- and
saline-challenged mice (Fig. 2). No
significant differences in airway responses were observed between the
saline-challenged mice of the different treatment groups.
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, A, B,
and C), at doses ranging from 12.5 to 50 mg/ml methacholine.
Interestingly, treatment with a combination of IL-12 and IL-18
significantly (p < 0.05) inhibited this airway
hyperresponsiveness, resulting in similar Penh values for both OVA- and
saline-challenged mice (Fig. 2D). Thus, OVA-challenged mice
demonstrate airway hyperresponsiveness compared with saline-challenged
mice, which can be completely inhibited by a combination of IL-12 and
IL-18 treatment, but not by treatment with IL-12 or IL-18 only. Bronchoalveolar lavage
The number of cells in the bronchoalveolar lavage was used as a measure for the infiltration of cells in the airways.
In mice treated with vehicle, IL-12, and the combination of IL-12 and
IL-18, the numbers of mononuclear cells in the bronchoalveolar lavage
were increased, although not statistically significant, in OVA- versus
saline-challenged mice. In IL-18-treated mice, the numbers of
mononuclear cells were significantly (p <
0.05) increased in OVA-challenged mice compared with vehicle-treated
OVA-challenged mice (Fig. 3C).
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A). The numbers of eosinophils between vehicle-,
IL-12-, or IL-18-treated OVA-challenged mice were not significantly
different. However, treatment with a combination of IL-12 and IL-18
almost completely inhibited (88%, p < 0.05) the
bronchoalveolar lavage eosinophilia in OVA-challenged mice (Fig. 3A) compared with vehicle-treated OVA-challenged mice.
The numbers of neutrophils in the bronchoalveolar lavage of
OVA-challenged mice treated with vehicle, IL-12, or IL-18 were
significantly (p < 0.01) increased when
compared with the corresponding saline-challenged mice (Fig. 3B). Treatment with a combination of IL-12 and IL-18 almost
completely inhibited (80%, p < 0.01) the neutrophils
in the lavage of OVA-challenged mice (Fig. 3B) compared with
vehicle-treated OVA-challenged mice. Therefore, OVA challenge induced
an eosinophilic and neutrophilic cellular infiltrate in the
bronchoalveolar lavage. This inflammation was almost completely
inhibited by treatment with a combination of IL-12 and IL-18, but not
by treatment with IL-12 or IL-18 only.
Cytokine production by Ag-stimulated thoracic lymph node and lung tissue cells
From each animal, the lung-draining lymph node cells and lung
tissue cells were isolated and stimulated with Ag in vitro for 5 days.
In previous studies, we have determined the optimal conditions for
Ag-stimulated cytokine production.3 In the supernatants of
these cultures, IFN-, IL-4, and IL-5 levels were measured.
Thoracic lymph node cells
Thoracic lymph node cells cultured in medium only produced
undetectable levels of cytokines (data not presented). In all treatment
groups, thoracic lymph node cells from saline-challenged mice produced
low levels of cytokines upon Ag-specific stimulation (Fig. 4). After repeated OVA inhalation,
thoracic lymph node cells from vehicle-treated mice produced low levels
of IFN- and significantly (both p < 0.01) higher
levels of IL-4 and IL-5 compared with cultures of saline-challenged
mice (Fig. 4), indicating differentiation into the Th2-like cell
population. Compared with these cultures, Ag-stimulated thoracic lymph
node cells of IL-12-treated mice produced comparable IFN- and IL-4
levels, but significantly (p < 0.05) increased
IL-5 levels. In lymph node cell cultures from IL-18-treated mice,
comparable levels of IL-4, IL-5, and IFN- were detected compared
with similar cultures of vehicle-treated mice. Treatment with a
combination of IL-12 and IL-18 resulted in significantly lower levels
of IL-4 (71% inhibition, p < 0.05), decreased levels
of IL-5 (43% inhibition), and increased levels of IFN- (187%
increase) compared with similar cultures of vehicle-treated mice,
indicating the presence of Th0-like cells.
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Lung tissue cells
In agreement with previous studies,3 only IL-5, but
not IFN- and IL-4, levels were detectable in Ag-stimulated lung cell
cultures.
In Ag-stimulated lung cell cultures from saline-challenged mice, only
low levels of IL-5 could be measured (Fig. 5); these levels did not differ between
the treatment groups. In all treatment groups, lung cell cultures from
OVA-challenged mice showed significantly increased IL-5 levels
(p < 0.05) compared with cultures from
saline-challenged mice. Treatment of mice with either IL-12 or IL-18
during OVA challenge did not alter the IL-5 levels in Ag-stimulated
lung cell cultures. However, Ag-stimulated lung cell cultures from
OVA-challenged mice treated with a combination of IL-12 and IL-18
showed a significantly decreased (76%, p < 0.05) IL-5
production.
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Stimulation of thoracic lymph nodes and lung cells in the presence of IL-12 and/or IL-18
To study the direct effect of IL-12 and IL-18 on the cytokine production by Th2-like cells, thoracic lymph node and lung cells from OVA-sensitized and OVA-challenged mice were Ag stimulated in the presence of IL-12 and/or IL-18. Different concentrations of IL-12 and IL-18 were used, and shown are the doses of IL-12 and IL-18, resulting in the maximal effects.
In both non- and Ag-stimulated thoracic lymph node and lung cell
cultures, the addition of IL-12 induced dose dependently low levels of
IFN- (respectively 4.1 ± 0.2 ng/ml and 0.9 ± 0.09 ng/ml)
(Fig. 6A) as compared with
polyclonal-stimulated cell cultures (respectively >8 ng/ml and
2.5 ± 0.05 ng/ml). Although IL-18 itself did not induce IFN-
production, it enhanced dose dependently the IL-12-induced IFN-
production in thoracic lymph node and lung cell cultures irrespective
of the stimulation.
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B).
In OVA-stimulated thoracic lymph node cell cultures, addition of IL-18
slightly reduced the IL-4 levels (28% inhibition). Addition of IL-12
alone did not alter the IL-4 levels in these cultures.
The IL-5 levels (Fig. 6C) in OVA-stimulated lung cell
cultures were slightly reduced by the addition of IL-12 (28%
inhibition). Addition of IL-18 did not alter the IL-5 levels in these
cultures. In contrast, the IL-5 levels in Ag-stimulated thoracic lymph
node cell cultures were not influenced by the addition of IL-12, but
slightly decreased by the addition of IL-18 (35% inhibition). No
synergistic effects of IL-12 and IL-18 were observed on the IL-4 and
IL-5 levels by Ag-stimulated thoracic lymph node and lung cell
cultures.
Thus, a combination of IL-12 and IL-18 synergistically induced IFN-
production in an Ag-independent manner, whereas no synergistic effect
on IL-4 and IL-5 levels was observed.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In literature, conflicting data exist on the role of IL-12 in secondary
IgE responses in mice. Similar to the present study, unaltered IgE
levels have been observed after treatment with IL-12 during the
challenge period (21, 22, 23), but others have demonstrated that IgE
responses could be inhibited using a similar dose of IL-12 (14). The
difference in Ag used in the latter study compared with the other
studies may account for this discrepancy. Interestingly, in our
experiments, the potentiation of the IgE levels could almost completely
be inhibited by treatment with a combination of IL-12 and IL-18. These
data are in accordance with a study by Yoshimoto and colleagues (14),
who suggested that the inhibition of IgE levels by IL-12 and IL-18 was
mediated via an IFN--dependent mechanism. In line with these data,
we and others have shown that treatment with IFN- during Ag
inhalation or during sensitization can inhibit the production of IgE in
mice (24, 25, 26). Furthermore, IFN- can also inhibit IgE production by
B cells in vitro (27).
In the present study, eosinophil and neutrophil infiltration was observed in the bronchoalveolar lavage after repeated OVA challenge. Infiltration of eosinophils in the airways is one of the characteristic features of allergic asthma. The eosinophils are thought to be responsible for the airway epithelial damage in patients with allergic asthma, which may result in the induction of airway hyperresponsiveness (28). In the present study, treatment with a combination of IL-12 and IL-18 during the challenge period was effective in almost completely inhibiting the eosinophilic and neutrophilic inflammation after OVA challenge. It is generally accepted that IL-5 is essential for the eosinophilic infiltration in the airways (16). In addition, depletion of CD4+ cells in mice could also prevent allergen-induced eosinophilic infiltration (29), indicating the role of Th2-like cells in this response. Interestingly, in our experiments, eosinophilic infiltration in OVA-challenged mice was accompanied with IL-5 production by Ag-stimulated lung cells, and both were inhibited in mice treated with a combination of IL-12 and IL-18. It can be speculated that IL-5 production by Ag-specific lung cells is responsible for the eosinophilic infiltration. Similarly, lung CD4+ cells from atopic patients produced high levels of IL-5 after allergen challenge (30). No altered eosinophilic and neutrophilic infiltration was observed after treatment with either IL-12 or IL-18 during the challenge period. This is in contrast with other studies, which showed that IL-12 treatment during the challenge period can inhibit airway eosinophilia (9, 21, 31, 32). In these studies, mice were treated with a 10 times higher dose of IL-12 than the present study, which probably explains the different results.
In patients with allergic asthma, it was shown that airway hyperresponsiveness correlates with disease severity (33), and is therefore a major feature of allergic asthma. In our experiments, OVA-challenged mice showed airway hyperresponsiveness that was effectively inhibited by treatment with a combination of IL-12 and IL-18, but not by treatment with either IL-12 or IL-18. Contrasting data exist on the effect of IL-12 on airway hyperresponsiveness during the challenge period. Similar to the present study, Sur and colleagues demonstrated that treatment with IL-12 during the challenge period resulted in unaltered airway hyperresponsiveness (23). In contrast, inhibition of the airway hyperresponsiveness was observed by others using the same dose of IL-12 (9, 21). As mentioned before, the dose of IL-12 in these studies was higher compared with the present study, which can partially explain the differences.
In previous studies, we have characterized the cytokine production by
Ag-stimulated T cells derived from the lungs and draining lymph nodes
in mice with and without airway symptoms.3 No Ag-specific T
cell responses were observed after sensitization in lung-draining lymph
nodes or lung tissue in the present study. However, after repeated OVA
challenges, Ag-stimulated lymph node cells produced IL-4 and IL-5. More
importantly, concomitant with development of airway eosinophilia,
Ag-stimulated lung cells produced only IL-5, but no IFN- or IL-4. In
previous studies (19), we showed that CD4+ cells are
responsible for the Ag-induced cytokine production in these cell
cultures, implicating the presence of Th2-like cells. Thus, airway
symptoms were observed concomitant with Th2-like cell development,
indicating an important role for Th2-like cells and the cytokines they
produce. In addition, in murine studies it was shown that IL-4 is
essential for IgE production (15), and that IL-5 is necessary for
eosinophilic infiltration in the airways (34). After treatment with a
combination of IL-12 and IL-18 in vivo, decreased IL-4 and IL-5 levels
and increased IFN- levels were observed by Ag-stimulated lymph node
cells. Ag-stimulated lung cells produced decreased levels of IL-5, but
still no IFN-. These data indicate that either the differentiation
of Ag-specific T cells to the Th2-like phenotype is prevented or that
the synthesis of Th2-like cytokines is inhibited. To examine the latter
explanation, the direct effects of IL-12 and IL-18 on Th2-like cells
derived from thoracic lymph node and lung cells were studied. Addition
of IL-12 induced IFN- production by thoracic lymph node and lung
cells from OVA-sensitized and OVA-challenged mice. The IL-12-induced
IFN- production was further enhanced by the addition of IL-18. These
results are in agreement with other in vitro studies (11, 12, 13, 14) in which
synergistic effects of IL-12 and IL-18 on IFN- production by T cells
were observed. However, opposite to the effects of in vivo treatment,
the combination of IL-12 and IL-18 in vitro does not synergistically
reduce the cytokine production by Th2-like cells. Therefore, it can be
suggested that in vivo treatment with a combination of IL-12 and IL-18
prevents the development of Th2-like cells rather than reducing the
cytokine production by Th2-like cells.
Treatment with either IL-12 or IL-18 did not result in an altered cytokine profile by Ag-stimulated lymphocytes. This is in contrast with a murine study of airway inflammation in which IL-12 treatment inhibited the IL-4 and IL-5 production by Ag-stimulated splenocytes (32). The high dosage of IL-12 used in this study might have contributed to the decreased cytokine production.
In the present study, we observed that treatment with a combination of
low doses of IL-12 and IL-18, but not treatment with either cytokine,
can inhibit airway inflammation symptoms. The mechanism of the
synergistic effect of IL-12 and IL-18 on the inhibition of the airway
inflammatory symptoms is still unclear, but several explanations can be
postulated. First, although IL-18 itself is unable to induce
differentiation to the Th1-like phenotype, it has been shown to enhance
IL-12-induced Th1 differentiation (12). In our experiments, the
relatively low dose of IL-12 used seems not sufficient to enhance the
development to Th1-like cells or to inhibit the development of Th2-like
cells. This could explain why treatments with either IL-12 or IL-18
were not capable of inhibiting the Th2-like cell development, while the
combined treatment was effective. Second, it has been shown that IL-18
can enhance the IL-12-induced IFN- production (11, 12, 13, 14). Previously,
we (24) and others have shown that treatment with IFN- can inhibit
IgE production (25, 26), and several airway inflammatory processes such
as airway hyperresponsiveness (17, 26) and eosinophilic infiltration
(31, 35, 36). Although IFN- has no effect on IL-4 and IL-5
production by T cells upon restimulation in vitro (24), it has been
shown to inhibit the proliferation of Th2-like cells, resulting in a
less pronounced Th2 skewing. However, using IFN- receptor-deficient
mice, it was shown that the inhibition of eosinophil influx by
treatment with IL-12 during the challenge period is independent of
IFN- (22). Thus, it remains possible that IFN--independent
mechanisms are involved in the effects of IL-12 and IL-18 in vivo.
Third, an explanation for the synergistic effect of IL-12 and IL-18 can
be found in the fact that IL-12 can up-regulate the expression of
IL-18R (13), and in this way stimulate the effects mediated by IL-18.
From the present study, we can conclude that the combination of low doses of IL-12 and IL-18 treatment can prevent Th2-like cell development concurrent with inhibition of airway symptoms. More knowledge is required to fully understand the synergistic mechanism of IL-12 and IL-18 in vivo.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Claudia L. Hofstra, Department of Pharmacology and Pathophysiology, Utrecht University, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands. E-mail address:
3 C. L. Hofstra, I. Van Ark, M. Kool, F. P. Nijkamp, and A. J. M. Van Oosterhout. Antigen-stimulated CD4+ cells produce IL-5, while lymph node CD4+ cells produce Th2 cytokines concomitant with airway eosinophilia and hyperresponsiveness. Submitted for publication.
Received for publication March 24, 1998. Accepted for publication June 23, 1998.
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  T. Yoshimoto, T. Yoshimoto, K. Yasuda, J. Mizuguchi, and K. Nakanishi IL-27 Suppresses Th2 Cell Development and Th2 Cytokines Production from Polarized Th2 Cells: A Novel Therapeutic Way for Th2-Mediated Allergic Inflammation J. Immunol., October 1, 2007; 179(7): 4415 - 4423. [Abstract] [Full Text] [PDF]
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 S. Matsubara, K. Takeda, T. Kodama, A. Joetham, N. Miyahara, T. Koya, C. H. Swasey, M. Okamoto, A. Dakhama, and E. W. Gelfand IL-2 and IL-18 Attenuation of Airway Hyperresponsiveness Requires STAT4, IFN-{gamma}, and Natural Killer Cells Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 324 - 332. [Abstract] [Full Text] [PDF]
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I. Meyts, P. W. Hellings, G. Hens, B. M. Vanaudenaerde, B. Verbinnen, H. Heremans, P. Matthys, D. M. Bullens, L. Overbergh, C. Mathieu, et al. IL-12 Contributes to Allergen-Induced Airway Inflammation in Experimental Asthma J. Immunol., November 1, 2006; 177(9): 6460 - 6470. [Abstract] [Full Text] [PDF]
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M. V. Lopez, S. K. Adris, A. I. Bravo, Y. Chernajovsky, and O. L. Podhajcer IL-12 and IL-10 Expression Synergize to Induce the Immune-Mediated Eradication of Established Colon and Mammary Tumors and Lung Metastasis J. Immunol., November 1, 2005; 175(9): 5885 - 5894. [Abstract] [Full Text] [PDF]
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I. P. Lewkowich, J. D. Rempel, and K. T. HayGlass Prevention of Allergen-Specific, Th2-Biased Immune Responses In Vivo: Role of Increased IL-12 and IL-18 Responsiveness J. Immunol., October 15, 2005; 175(8): 4956 - 4962. [Abstract] [Full Text] [PDF]
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M. Patel, D. Xu, P. Kewin, B. Choo-Kang, C. McSharry, N. C. Thomson, and F. Y. Liew TLR2 Agonist Ameliorates Established Allergic Airway Inflammation by Promoting Th1 Response and Not via Regulatory T Cells J. Immunol., June 15, 2005; 174(12): 7558 - 7563. [Abstract] [Full Text] [PDF]
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S.-Z. Wang, Y.-X. Bao, C. L. Rosenberger, Y. Tesfaigzi, J. M. Stark, and K. S. Harrod IL-12p40 and IL-18 Modulate Inflammatory and Immune Responses to Respiratory Syncytial Virus Infection J. Immunol., September 15, 2004; 173(6): 4040 - 4049. [Abstract] [Full Text] [PDF]
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C. Taube, J. A. Nick, B. Siegmund, C. Duez, K. Takeda, Y.-H. Rha, J.-W. Park, A. Joetham, K. Poch, A. Dakhama, et al. Inhibition of Early Airway Neutrophilia Does Not Affect Development of Airway Hyperresponsiveness Am. J. Respir. Cell Mol. Biol., June 1, 2004; 30(6): 837 - 843. [Abstract] [Full Text] [PDF]
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V. Smart, P. S. Foster, M. E. Rothenberg, T. J. V. Higgins, and S. P. Hogan A Plant-Based Allergy Vaccine Suppresses Experimental Asthma Via an IFN-{gamma} and CD4+CD45RBlow T Cell-Dependent Mechanism J. Immunol., August 15, 2003; 171(4): 2116 - 2126. [Abstract] [Full Text] [PDF]
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J.C. Kips, G.P. Anderson, J.J. Fredberg, U. Herz, M.D. Inman, M. Jordana, D.M. Kemeny, J. Lotvall, R.A. Pauwels, C.G. Plopper, et al. Murine models of asthma Eur. Respir. J., August 1, 2003; 22(2): 374 - 382. [Abstract] [Full Text] [PDF]
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