| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Division of Immunology and Allergy, Department of Pediatrics,
Division of Oncology, Department of Medicine, and
Department of Pathology, Stanford University Medical Center, Stanford, CA 94305
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, was administered into the respiratory
tract as cDNA in a replication-deficient adenovirus (Adv). Treatment of
OVA-sensitized mice with the IL-18-expressing Adv reduced
allergen-specific IL-4 production, airway eosinophilia, and mucus
production, increased IFN- production, and prevented the development
of AHR. The effects of the IL-18 Adv treatment were dependent on the
presence of IFN- and IL-12. Moreover, administration of the IL-18
Adv to mice with established AHR greatly reduced AHR and IL-4
production and increased IFN- production. These results demonstrate
that IL-18, when administered by Adv into the respiratory tract,
effectively reduces AHR and replaces an established Th2-biased immune
response with a Th1-biased response. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
There has been much interest focused on the role of allergen-specific
Th1 cells, producing high levels of IFN-, as regulators of allergic
inflammation that can down-modulate Th2-biased immune responses in the
respiratory tract, thereby protecting against the symptoms of allergy
and asthma. This idea is supported by a large number of experimental
studies. For example, Th1 cells cross-regulate Th2 cells by inhibiting
the development and proliferation of Th2 cells, and IgE production is
reciprocally regulated by IL-4 and IFN- (6). In
addition, long-term allergen-specific T cell clones from the peripheral
blood of nonallergic individuals have been shown to produce Th1
cytokines (7, 8, 9, 10, 11). Moreover, we and others have shown that
individuals predisposed toward the production of Th1 cytokines (i.e.,
patients with Mycobacteria tuberculosis infection
(12) or those with autoimmune disease
(13, 14, 15)) have a reduced likelihood of developing allergic
disease. Thus, enhanced IFN- production is associated with
protection from the development of allergic disorders and asthma.
IL-12 is the most potent inducer of IFN- production and of Th1 cell
differentiation, but IFN- production is enhanced an additional
10-fold by the presence of IL-18 (16). In addition, the
combination of IL-12 with IL-18 reduces IgE synthesis and induces
IFN- production in B cells (17). Moreover, IL-18,
induced by CpG motifs in plasmids (18) or by heat-killed
Listeria monocytogenes (19), was involved in
limiting the development of Th2-biased immune responses and preventing
allergen-induced AHR. Because of these characteristics, we hypothesized
that administration of IL-18 directly into the airways of mice would
inhibit Th2-dominated immune responses and allergen-induced AHR.
Therefore, we administered IL-18 intranasally (i.n.) as cDNA in a
nonreplicating adenovirus (Adv) and examined the capacity of IL-18 to
attenuate OVA-induced AHR.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
BALB/c ByJ mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Animals were used between 6 and 10 wk of age and were age and sex matched within each experiment. The Stanford University Committee on Animal Welfare approved all animal protocols.
Cell lines and bacteria
293A is a subclone of 293, the human embryonic kidney cell line that stably expresses the human Adv type 5 DNA (Quantum Biotech, Quebec, Canada). BJ5183 is a recombination-proficient strain of bacteria (kindly provided by M. Mehtali, Transgéne, Strasbourg, France).
Monoclonal Abs
mAbs were purified from ascites fluid by ammonium sulfate
precipitation and ion-exchange chromatography. We used the following
hybridomas: R46A2 (anti-IFN- mAb), obtained from American Type
Culture Collection (Manassas, VA); XMG1.2 (anti-IFN- mAb),
generously provided by Dr. T. Mosmann (University of Rochester,
Rochester, NY); BVD4-1D11 (anti-IL-4) and BVD6-24G2 (anti-IL-4
mAb), obtained from Dr. M. Howard, (Corixa, Redwood City, CA); C17.8
(anti-IL-12 mAb), obtained from Dr. G. Trinchieri (Philadelphia,
PA); and EM95 (rat anti-mouse IgE mAb), kindly provided by Dr. R.
Coffman, (DNAX Research Institute, Palo Alto, CA). Anti-OVA mAbs and
biotinylated anti-OVA mAb were produced as described previously
(20). Anti-38C13 Id mAb 4G10 (rat IgG2a; Ref.
21) was used as control.
Generation of recombinant Adv
Cloning and recombination. Murine IL-18 cDNA was cloned into Adv transfer vector pXCJ-1 CMV/pA (pXCJ-IL18). pXCJ-1 CMV/pA is a mammalian expression vector that contains adenoviral sequences corresponding to 0–452 bp and 3328–5789 bp of Ad 5 genome flanking the multiple cloning site (kindly provided by I. Verma, Salk Institute, San Diego, CA). The Adv-derived sequences facilitate the homologous recombination of IL-18 into the E1 locus of the Adv type 5 genome, encoded on the plasmid pTG3652 (kindly provided by M. Mehtali, Transgéne; Ref. 22).
Recombination of pXCJ-IL18 and pTG3652 plasmids in bacteria was performed as reported previously (22, 23), with slight modification. Linearized pXCJ-IL-18 was cotransformed with ClaI-linearized pTG3652 into recombination-proficient BJ5183 cells. Recombination was confirmed by restriction digest mapping of plasmid DNA from bacterial colonies. The recombined TG3652-IL18 plasmid was retransformed into XL1Blue cells for large-scale plasmid purification.
Transfection with recombinant adenoviral DNA plasmid. 293A cells were transfected with 1 µg of PacI-linearized TG3652-IL18 with Lipofectamine Plus (Life Technologies, Gaithersburg, MD). One day after transfection, transfected cells were immobilized with medium containing 0.5% agarose and monitored for viral plaque formation. Viral plaques were isolated and used to generate primary and amplified viral stocks. Correct IL-18:Adv clones were confirmed by PCR analysis.
Adv purification. Purification of Adv was done according to published reports with slight modification (24). 293A cells were infected with amplified viral stock diluted into sterile PBS. When cytopathic effects were apparent, infected cells were harvested, resuspended in TE, and lysed by three freeze/thaw cycles. Viral lysate was mixed with saturated cesium chloride (in Tris-EDTA buffer, pH 8.0) at v/v ratio of 3.1 ml of viral supernatant to 1.8 ml of CsCl. Virus/CsCl mixture was spun in a Beckman ultracentrifuge (Beckman Coulter, Fullerton, CA) at 35,000 rpm, 4°C, for 16–20 h. The viral band was extracted and dialyzed extensively against PBS containing 10% glycerol at 4°C. Viral titer was determined by plaque titration in 293 cells. Aliquots of purified virus was stored frozen at -70°C. A control Adv encoding luciferase (lucif:Adv; Ref. 25) was purified, titered, and stored by identical procedures.
IL-18 production. To confirm that IL-18:Adv produced IL-18 protein, HeLa cells were infected with 2, 20, or 200 multiplicity of infection (MOI) of virus. When cytopathic effects were apparent, supernatants from the transfected cells were harvested, and production of adenovirally expressed IL-18 protein was measured by SDS-PAGE and Western blotting with a goat anti-mouse IL-18 Ab at 0.5 µg/ml (Research Diagnostics, Flanders, NJ).
Immunization protocols
Protocol 1: prevention of AHR.
To determine whether IL-18:Adv could prevent the development of AHR,
BALB/c mice were given the virus i.n. while the mice were being
sensitized to OVA by an established protocol for the induction of AHR
in BALB/c mice (Ref. 26 ; see Fig. 1
C). Briefly,
OVA (50 µg) adsorbed to 2 mg of aluminum potassium sulfate (alum) was
administered i.p. on day 0. On day 7, 1.0 x
108 MOI of IL-18:Adv in 0.9% normal saline
(n.s.) was administered i.n., followed by 50 µg of OVA in 50 µl of
0.9% n.s. given i.n. on days 10, 11, and 12. Control mice received
i.p. injections of alum alone and/or 1.0 x
108 MOI of lucif:Adv in 0.9% n.s. i.n., and OVA
i.n. as described above. One day after the last i.n. challenge (day
13), AHR was measured in conscious mice after inhalation of increasing
concentrations of methacholine (see below). Two days after the last
challenge, mice were bled and then sacrificed, lungs were removed, and
bronchial lymph nodes were isolated for in vitro culture.
|
). A total of 50
µg of OVA adsorbed to alum was administered i.p. on day 0. On day 7,
50 µg OVA in 50 µl of 0.9% n.s. was administered i.n. On day 14,
1.0 x 108 MOI of IL-18:Adv in 0.9% n.s.
then was administered i.n. On days 17, 18, and 19, the mice were
boosted again with OVA i.n., and AHR was measured 1 day later (day 20).
On day 21, mice were bled and then sacrificed as described for
protocol 1. Treatment of mice with anti-cytokine and depletion mAb
BALB/c mice undergoing treatment to prevent AHR were injected
i.p. with 1 mg of mAb XMG1.2 (for IFN- depletion), with 1 mg of mAb
C17.8 (for IL-12 depletion) or 1 mg of mAb 4G10 (control mAb) every 4
days, starting on day 6, 1 day before administration of the virus. The
Abs were present throughout the course of immunization and assessment
of AHR.
Measurement of AHR
AHR was assessed by methacholine-induced airflow obstruction from conscious mice placed in a whole-body plethysmograph (model PLY 3211; Buxco Electronics, Troy, NY), as described previously (26).
Determination of lung histology
Animals were sacrificed by CO2 asphyxiation. The lungs were removed, washed in PBS, inflated, fixed in 10% neutral buffered formalin, embedded in paraffin wax, sectioned at 5-µm thickness, and stained with hematoxylin and eosin (H&E).
Restimulation of lymph node cells in vitro
Cells (3.0 x 106 cells/well in a
24-well plate for cytokine ELISAs or 5.0 x
105 cells/well in a 96-well plate for
proliferation) isolated from bronchial lymph nodes were restimulated in
vitro in the presence or absence of 100 µg/ml OVA. For ELISAs,
supernatants were harvested after 2 days for determination of IL-12
levels, and after 4 days for determination of IL-4, IL-5, IL-13, and
IFN- levels. For proliferation, cultures were incubated for 4 days
and pulsed with 1 µCi of [3H]thymidine for
the last 18 h.
Cytokine ELISAs
ELISAs were performed as described previously (27).
The mAb pairs used were as follows, listed by capture/biotinylated
detection mAb: IFN-, R4-6A2/XMG1.2; IL-4, 11B11/BVD6-24G2; IL-12,
C17.8/C15.6; IL-5, TRFK5/TRFK4; IL-13 mouse Abs (R&D Biosystems,
Minneapolis, MN); IL-18, OptEIA mouse ELISA Kit (BD PharMingen, San
Diego, CA).
OVA-specific IgE assay
Mice were bled at the time of sacrifice, and OVA-specific IgE was determined by using a modified Ag-specific ELISA as described previously (28).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To express IL-18 in vivo, a nonreplicating Adv encoding the open
reading frame of mature murine IL-18 under control of the CMV promoter
was made and termed IL-18:Adv. To confirm that IL-18:Adv produced the
IL-18 protein, HeLa cells were infected with varying amounts of virus,
supernatants from the transfected cells were harvested, and production
of Adv-expressed IL-18 protein was determined by Western blotting. Fig. 1A shows that a MOI of 20
resulted in a faint but detectable signal, whereas a MOI of 200
resulted in a strong signal of the appropriate size by Western
blotting, indicating that the IL-18 Adv construct produced the IL-18
protein.
To demonstrate that IL-18 protein was produced in vivo after i.n.
administration of the IL-18:Adv, we collected serum and
bronchioalveolar (BAL) fluid serially after Adv administration. Fig. 1B shows that IL-18 expression peaked on day 2 in BAL fluid
and slowly declined over the next week. In contrast, no IL-18 activity
was detected in the blood at any time point, and no IL-18 was detected
in BAL fluid when the control luciferase-expressing virus (lucif:Adv)
was administered, indicating that the Adv itself did not induce
detectable IL-18 production.
IL-18 can prevent the development of AHR in mice
The IL-18:Adv next was tested in vivo for its ability to inhibit
AHR in a murine model of asthma. AHR was induced in mice by i.p.
administration of OVA followed by i.n. challenge with OVA on days 10,
11, and 12. On day 7, mice received i.n. virus (either IL-18:Adv or the
control virus, lucif:Adv; Fig. 1C, protocol 1). Fig. 2A demonstrates that mice
sensitized with OVA and challenged with i.n. OVA developed significant
AHR, elicited with increasing concentration of methacholine. However,
OVA-sensitized and -challenged mice that were given i.n. IL-18:Adv had
significantly reduced levels of AHR. The effect was IL-18-specific, as
lucif:Adv failed to reduce AHR. Furthermore, the IL-18 and luciferase
viruses by themselves did not have a measurable effect on AHR. The peak
Penh values for the virally treated mice that were not sensitized with
i.p. OVA were not significantly different from that of mice that just
received i.n. OVA (data not shown), although the baseline Penh levels
of these mice treated with virus were slightly elevated.
|
IL-18 is a cytokine that has been shown to be a potent inducer of
IFN- in CD4 and CD8 cells. To determine whether i.n. administration
of IL-18:Adv induced a Th1-like response in the lungs, the mice were
sacrificed, and bronchial lymph node cells were removed and stimulated
in vitro with OVA. Fig. 2B shows that lymph node cells from
mice that were sensitized with OVA i.p. and challenged with i.n. OVA
proliferated in response to OVA. The bronchial lymph node cells of the
mice that received i.n. OVA without i.p. sensitization to OVA but were
treated with the IL-18:Adv also proliferated in response to OVA.
Examination of cytokine levels in culture supernatant derived from mice
that were sensitized and challenged with OVA showed that IL-18:Adv
significantly decreased IL-4, IL-5, and IL-13 production, whereas
lucif:Adv had little effect. IL-18:Adv also significantly increased
OVA-induced IFN- production in mice that were sensitized and
challenged with OVA and in mice that received only i.n. OVA.
Effect of IL-18:Adv on lung histology
Lung sections were taken from mice in all treatment groups, fixed
in formalin, and stained with H&E. Lungs from OVA-sensitized and
-challenged mice treated with lucif:Adv contained intense inflammation,
with significant eosinophilia and increased mucus production, as
evidenced by the distended epithelial cells that are engorged with
large quantities of mucin (Fig. 3A). Similar results were
observed in sections from mice sensitized with OVA i.p. and challenged
with i.n. OVA (data not shown). Lungs from OVA-sensitized and
-challenged mice treated with IL-18:Adv contained less inflammatory
cells and less eosinophils (Fig. 3B). In addition, IL-18:Adv
greatly reduced production and secretion of mucus, as reflected by the
normal size of the epithelial cells, and reduction in intracytoplasmic
and intraluminal mucin. However, a mononuclear infiltrate remained,
presumably because of a Th1-biased response, despite the fact that
airway reactivity in response to methacholine was nearly absent. Mice
treated with lucif:Adv that received only i.n. OVA (but not i.p. OVA)
had essentially normal histology (Fig. 3C). In contrast,
mice treated with IL-18:Adv that received only i.n. OVA (but not i.p.
OVA) had peribronchial infiltrates, consisting of mononuclear cells,
with virtually no mucus production or eosinophils (Fig. 3D),
as did mice that received IL-18:Adv alone (without i.p. or i.n. OVA;
data not shown). This indicated that IL-18:Adv itself induced a
discernible Th1-like inflammation in the lungs, characterized by
mononuclear but not eosinophilic cell infiltrates.
|
and IL-12
To investigate the mechanism by which administration of IL-18:Adv
affected OVA-induced responses, we administered anti-IFN- mAb or
anti-IL-12 mAb during the immunization protocol. As expected,
OVA-sensitized and -challenged mice that were treated with IL-18:Adv
alone or in combination with the control mAb had significantly lower
AHR when compared with OVA-sensitized and -challenged mice (Fig. 4A). However, treatment with
the anti-IFN- or anti-IL-12 mAb reversed the inhibition of
AHR conferred by IL-18:Adv (Fig. 4A). Treatment of
OVA-sensitized and -challenged mice with either the anti-IFN- or
the anti-IL-12 mAb had no significant effect on AHR (data not
shown). Thus, inhibition of AHR by IL-18:Adv was dependent on both
IFN- and IL-12 production.
|
mAb or anti-IL-12 mAb
blocks induction of a Th1 response by IL-18:Adv
Because treatment of mice with anti-IFN- or anti-IL-12
mAb blocked the reduction of AHR by IL-18:Adv, we asked if the Th1
response elicited by IL-18:Adv also was blocked. Bronchial lymph node
cells from the groups that had high AHR produced high amounts of IL-4
on restimulation with OVA in vitro, whereas cells from the mice that
received virus alone or virus plus the control Ab produced
significantly lower levels of IL-4 (Fig. 4B).
Coadministration of anti-IFN- or anti-IL-12 mAb in the
IL18:Adv-treated mice reversed the ability of IL-18:Adv to reduce IL-4
production. The blocking Abs also significantly reduced OVA-induced
IFN- production induced by IL-18:Adv in bronchial lymph node cells,
though the effect of the anti-IL-12 mAb was slightly less than that
of the anti-IFN- mAb. Examination of IL-12 levels in
supernatants of bronchial lymph node cells showed that administration
of IL-18:Adv significantly increased the levels of IL-12 over that seen
in OVA-sensitized mice that did not receive IL-18:Adv. Again,
administration of blocking Abs reduced the observed levels of IL-12.
Anti-IL-12 mAb reduced IL-12 levels to that of OVA-sensitized mice,
whereas anti-IFN- mAb reduced IL-12 by 
50%, whereas
treatment with control Ab had no effect on cytokine production.
IL-18:Adv reverses established AHR in mice
To determine whether IL18:Adv could reverse established AHR in
addition to inhibiting the development of AHR, mice were exposed to
virus after exposure of the mice to i.n. OVA, which induces AHR, as we
have shown previously (19 ; Fig. 1C, protocol
2). OVA-sensitized and -challenged mice that were subsequently given
i.n. IL-18:Adv had significantly reduced levels of AHR when compared
with OVA-sensitized and -challenged mice that did not receive virus
(Fig. 5A) or that were given
lucif:Adv (data not shown).
|
To determine whether i.n. administration of IL-18:Adv also
reversed the Th2 response associated with the established AHR, the mice
were sacrificed 1 day after measurement of AHR, and bronchial lymph
node cells were isolated and cultured in the presence or absence of
OVA. Lymph node cells from OVA-sensitized and -challenged mice
proliferated in vitro to a similar degree whether they did or did not
receive IL18:Adv (Fig. 5B). In addition, lymph node cells
from mice that received the IL-18:Adv and OVA i.n. without prior i.p.
sensitization also proliferated to OVA in vitro, suggesting that
IL-18:Adv acted as an adjuvant. Administration of IL-18:Adv to
OVA-sensitized and -challenged mice significantly decreased levels of
IL-4 production by bronchial lymph node cells when compared with the
OVA-sensitized mice that did not receive IL-18:Adv (Fig. 5B). Additionally, IL-18:Adv significantly increased
OVA-induced IFN- production in mice sensitized and challenged with
OVA. Mice that received i.n. OVA plus IL18:Adv without prior i.p.
sensitization produced little or no IL-4, but produced high levels of
IFN-.
Effect of IL-18:Adv on lung histology when established AHR is reversed
On the day of sacrifice, lung histology also was examined. Mice
sensitized and challenged with OVA showed evidence of a vigorous Th2
response, with intense inflammation, mucus production, and eosinophilia
(Fig. 6A). However, treatment
of OVA-sensitized and -challenged mice with IL-18:Adv (Fig. 6B) greatly reduced airway inflammation, mucus production,
and eosinophilia, but left a mononuclear cell infiltrate in the
peribronchiolar and perivascular spaces. Treatment of mice with
lucif:Adv that only received i.n. OVA had essentially normal lung
histology (Fig. 6C). The lung histology of mice that only
received i.n. OVA and were treated with IL-18:Adv (Fig. 6D)
had a mild inflammatory infiltrate composed mostly of mononuclear
cells, with little mucus production or tissue eosinophilia.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
while reducing
production of IL-4. In the lungs, IL-18:Adv reduced airway eosinophilia
and goblet cell hyperplasia, but it induced a Th1-biased mononuclear
cell inflammatory infiltrate in the airways. The reversal of
established AHR and generation of the Th1-biased inflammatory
infiltrate was primarily attributable to the presence of IL-18, because
administration of the lucif:Adv control construct had no effect on
airway inflammation in the presence or absence of OVA. These results
indicate that administration of IL-18 into the lungs of mice with an
Adv construct can effectively down-modulate established Th2-dominated
inflammatory responses in the respiratory tract. Previously published studies examining the role of IL-18 in allergen-induced AHR responses have generated contradictory results. For example, consistent with the idea that IL-18 suppresses Th2-related immune responses, the induction of allergen-induced airway eosinophilia was greatly increased in IL-18 knockout mice compared with that in control mice (29). Similarly, administration of IL-12 and IL-18 together inhibited the development of AHR, though neither alone inhibited AHR (30). In contrast, administration of IL-18 protein alone in very large quantities (7–20 µg/mouse, i.p.) in the absence of IL-12 paradoxically accentuated the recruitment of eosinophils into the airways (31) and enhanced allergic sensitization and IgE production (32).
The deleterious effects of IL-18 on AHR in some experimental systems
may have been attributable to induction of IL-13 and eotaxin production
by IL-18 (29, 31), resulting in the provocation rather
than protection against AHR. IL-18, like IL-12, possesses pleomorphic
activities that may either enhance or inhibit Th2-biased immune
responses (33). In the absence of IFN-, IL-18 induces
high levels of IL-13 (34), but in the presence of IFN-,
IL-18, can greatly amplify Th1-biased immune responses, particularly
because IL-18 induces IL-12 receptor expression (35).
Moreover, because IL-12 induces IL-18 receptor expression on T cells,
and because IL-18R is induced on Th1 but not Th2 cells
(36), IL-12 and IL-18 together are synergistic in
enhancing IFN- production (37), which then inhibits
Ag-induced AHR. Thus, the specific effects of IL-18 depend on the
circumstances of the administration (with or without IL-12) or on the
dose administered. Similar paradoxical effects have been associated
with IL-12, which under some circumstances, can enhance IL-4, IL-10,
and IgE production (38, 39, 40).
In our studies, it is likely that the salutary effects on AHR that we
observed with IL-18 administration were in part because of the
induction of IFN- and IL-12 by Adv components of our vector. Gene
transfer of IFN- into the airways inhibits the induction of AHR and
airway eosinophilia (41), and IL-12, in combination with
IFN- and IL-18 induces Th1 responses and inhibits the development of
AHR (30, 37), though neither alone has been shown to
reverse established AHR. In our experiments, anti-IFN- and
anti-IL-12 mAb both blocked the capacity of IL-18:Adv to prevent
the development of AHR, indicating that IL-18:Adv induced significant
quantities of both cytokines. In addition, administration of the
control lucif:Adv induced low levels of IL-12 production (data not
shown), and in some experiments, treatment with anti-IL-12 mAb
exacerbated the AHR in OVA-sensitized mice, resulting in more severe
AHR than that observed in OVA-sensitized controls (data not shown).
Thus, the Adv vector itself induced production of IL-12 and IFN-,
which synergized with IL-18 and contributed to the reduction in
AHR.
Our current experiments extend previous studies demonstrating that
coadministration of IL-18 and IL-12 inhibits the development of AHR
(30) and show for the first time that IL-18 can also
reverse established AHR. Although large doses of IL-18 protein in the
absence of IL-12 may exacerbate AHR (32), IL-18 expression
in the presence of IL-12 in our studies was clearly effective in
down-modulating established AHR. Our results also are consistent with
the adjuvant effects of agents that induce IL-18, such as CpG motifs
(42) and L. monocytogenes (19).
However, our studies more directly demonstrate that IL-18 is a primary
agent that exhibits antiasthma activity, and that IL-18 can reverse
ongoing disease, which is the major goal of therapeutic interventions
for asthma. The effects of IL-18:Adv when administered i.n. was
predominantly local, such that greatest effects were observed in the
lung and bronchial lymph nodes, and minimal effect was observed in the
spleen and on serum OVA-specific IgE levels (data not shown).
Administration of IL-18:Adv s.c. in the footpads also resulted in
enhanced IFN- production in the draining lymph nodes, but there was
no discernable inhibitory effect on AHR (data not shown). Thus,
administration of IL-18:Adv into the respiratory tract greatly enhanced
its inhibitory effects on AHR, or alternatively, IL-18:Adv administered
i.n. limited the recruitment of inflammatory cells into the lungs or
had direct effects on the cells recruited into the lung.
Although IL-18:Adv reversed established AHR and its associated Th2
response, this form of therapy did not result in a normal lung with
absence of pulmonary inflammation. Rather, IL-18:Adv induced a Th1-like
response in the lung, although this was not associated with increased
airway reactivity to methacholine. This response also could be observed
in naive mice treated with IL-18:Adv and in mice treated with i.n. OVA
and IL-18:Adv, and was characterized by production of high levels of
IFN- without detectable IL-4, and by recruitment of mononuclear
cells into the lung. This Th1-biased immune response appears to be
effective in reversing even ongoing AHR, but its long-term effects on
pulmonary physiology is not yet clear. It is possible that such
pulmonary Th1 responses are down-modulated rapidly, perhaps by the
presence of CD8 T cells or the induction of IL-10, and therefore
pulmonary inflammation might resolve without pulmonary scarring,
particularly when Ag is cleared from the lung. In contrast, there is
evidence to suggest that pure Th1 responses may adversely affect airway
inflammation and AHR (26, 43, 44, 45) by in fact enhancing
atopic inflammation. Nevertheless, in our studies, a single dose of
i.n. IL-18:Adv consistently reversed ongoing AHR, even when mice were
hyperimmunized with OVA in alum. These results suggest that cytokine
based, allergen-specific immunotherapies with IL-18 may be effective in
treating allergic diseases and asthma. Currently, conventional allergen
immunotherapy, performed by the s.c. injection of increasing doses of
allergen, is the only currently available therapy that alters the
underlying pathologic allergen-specific Th2-driven responses, resulting
in clinical tolerance to subsequent allergen exposure (10, 46). Our results leads us to speculate that IL-18, in
conjunction with IL-12, may serve as an effective adjuvant to enhance
the efficiency of allergen-based immunotherapies, which might be
effective in reversing the symptoms of asthma and allergy.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Dale T. Umetsu, Department of Pediatrics, Room G309, Stanford University, Stanford, CA 94305-5208.
3 Abbreviations used in this paper: AHR, airway hyperreactivity; Adv, adenovirus; MOI, multiplicity of infection; i.n., intranasal; alum, aluminum potassium sulfate; n.s., normal saline; BAL, bronchioalveolar lavage; H&E, hematoxylin and eosin.
Received for publication July 24, 2000. Accepted for publication March 8, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
secretion in neonates and subsequent atopy. Lancet 344:983.[Medline]
production by T cells. Nature 378:88.[Medline]
production from activated B cells. Proc. Natl. Acad. Sci. USA 94:3948.-inducing factor (IL-18) increases allergic sensitization, serum IgE, Th2 cytokines, and airway eosinophilia in a mouse model of allergic asthma. J. Immunol. 164:2701. production by resting T cells is IL-12 dependent and is mediated by up-regulation of the IL-12 receptor 
2 subunit. Eur. J. Immunol. 30:1113.[Medline]
production from CD3intIL- 2R+ T cells. J. Immunol. 161:1483. gene transfer inhibits pulmonary allergic responses in mice. J. Immunol. 157:3216.[Abstract]
production in cells from allergic individuals and inhibit IgE synthesis in vitro. Eur. J. Immunol. 29:2344.[Medline]
1 reverse allergen-induced airway hyperreactivity and inflammation. J. Clin. Invest. 105:61.[Medline]
messenger RNA expression of peripheral T lymphocytes. Immunology 87:593.[Medline]This article has been cited by other articles:
|
|
 |
|
  J. C. Horvat, K. W. Beagley, M. A. Wade, J. A. Preston, N. G. Hansbro, D. K. Hickey, G. E. Kaiko, P. G. Gibson, P. S. Foster, and P. M. Hansbro Neonatal Chlamydial Infection Induces Mixed T-Cell Responses That Drive Allergic Airway Disease Am. J. Respir. Crit. Care Med., September 15, 2007; 176(6): 556 - 564. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. W. Hellings, A. Kasran, D. Bullens, L. Overbergh, C. Mathieu, H. Heremans, P. Matthys, L. Boon, M. Jorissen, and J. L. Ceuppens IL-10- and IL-12-Independent Down-Regulation of Allergic Sensitization by Stimulation of CD40 Signaling J. Immunol., October 15, 2006; 177(8): 5138 - 5144. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Weigt, C. Nassenstein, T. Tschernig, P. F. Muhlradt, N. Krug, and A. Braun Efficacy of Macrophage-activating Lipopeptide-2 Combined with Interferon-{gamma} in a Murine Asthma Model Am. J. Respir. Crit. Care Med., September 1, 2005; 172(5): 566 - 572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. T. Ameredes, R. Zamora, J. M. Sethi, H.-L. Liu, L. K. Kohut, A. L. Gligonic, A. M. K. Choi, and W. J. Calhoun Alterations in Nitric Oxide and Cytokine Production with Airway Inflammation in the Absence of IL-10 J. Immunol., July 15, 2005; 175(2): 1206 - 1213. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Arora, Y. Hernandez, J. R. Erb-Downward, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Role of IFN-{gamma} in Regulating T2 Immunity and the Development of Alternatively Activated Macrophages during Allergic Bronchopulmonary Mycosis J. Immunol., May 15, 2005; 174(10): 6346 - 6356. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Xu, P. W. Park, F. Kheradmand, and D. B. Corry Endogenous Attenuation of Allergic Lung Inflammation by Syndecan-1 J. Immunol., May 1, 2005; 174(9): 5758 - 5765. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. de Nooijer, J.H. von der Thusen, C.J.N. Verkleij, J. Kuiper, J.W. Jukema, E.E. van der Wall, Th.J.C. van Berkel, and E.A.L. Biessen Overexpression of IL-18 Decreases Intimal Collagen Content and Promotes a Vulnerable Plaque Phenotype in Apolipoprotein-E-Deficient Mice Arterioscler Thromb Vasc Biol, December 1, 2004; 24(12): 2313 - 2319. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 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]
|
|
|
|
 |
|
 J. A. Gracie, S. E. Robertson, and I. B. McInnes Interleukin-18 J. Leukoc. Biol., February 1, 2003; 73(2): 213 - 224. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) given i.n. On the indicated day, BAL
was performed on the mice, and IL-18 levels were measured in the BAL
fluid by ELISA. C, Immunization schemes for prevention
of AHR and reversal of AHR established AHR with IL-18:Adv (see
Materials and Methods for details). Protocol 1 was used
to determine whether IL-18:Adv can prevent the development of AHR in
sensitized mice. This protocol was also used to examine the role IL-12
and IFN-
),
or medium supplemented with 100 µg/ml OVA (
). Proliferation then
was determined by [3H]thymidine incorporation, and IL-4,
IL-5, IL-13, and IFN-
