| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Divisions of
*
Immunology and Cell Biology and
Biochemistry and Molecular Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
but not
by any obvious reduction in the secretion of either IL-4 or IL-5, nor
by major changes in the IgG1 and IgE OVA-specific serum Ig profiles in
the mutants. The markedly enhanced production of IFN- in
NOS2-/- mice was apparently responsible for the
suppression of both eosinophilia and disease, as in vivo depletion of
this factor restored allergic pathology in these animals. Our data
indicate that NOS2 promotes allergic inflammation in airways via
down-regulation of IFN- activity and suggest that inhibitors of this
molecule may represent a worthwhile therapeutic strategy for allergic
diseases including asthma. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and IFN-, or microbial
products such as LPSs (5), in which it catalyzes the production of much
higher levels of NO mediating widespread antimicrobial effects (2).
Normally, resting cells do not express NOS2 unless triggered by
appropriate stimuli (6); however, cells such as macrophages, epithelial
cells, vascular smooth muscle, endothelial cells, T cells, and
eosinophils have the potential to express NOS2 when stimulated in this
manner (7, 8, 9). There is increasing evidence that NO also plays a role in the regulation of airways function in both health and disease. As in other tissues, both constitutive and inducible isoforms of NOS have been identified in lungs, with the latter implicated mainly in defense against infection and during airways inflammation (10). NO is produced by a variety of cell types in the respiratory tract (11, 12). It has been reported that asthmatic patients have increased expression of NOS2 in epithelial cells lining their airways (13, 14) and elevated levels of exhaled NO (15), while allergen-induced airway obstruction in a guinea pig model of allergic airway disease was also associated with high levels of exhaled NO (16). In addition, chemical inhibition of NO activity has recently been shown to suppress pulmonary eosinophilic inflammation in mice, although this was apparently not due to inhibition of NOS2 function (17).
Asthma is a chronic airways disease characterized by inflammation,
airway hyperreactivity (AHR), and reversible airway obstruction (18),
and it is likely that NO influences these pathophysiological events.
Pulmonary inflammation is thought to play a critical role in the
pathogenesis of asthma (18), with strong correlations between disease,
the local expression of Th2-type cytokines (including IL-4 and IL-5) by
lung T cells, and the presence of inflammatory cells, particularly
eosinophils, in blood and airways (19, 20). It has recently been
proposed that NO from airway epithelial cells, and possibly macrophages
and Th1 cells, promotes asthmatic inflammation via down-regulation of
Th1 cell-derived IFN- with concomitant up-regulation of Th2 cell
numbers and resultant increased local expression of IL-4 and IL-5 (21).
In addition, NO is a bronchodilator (22, 23) and a potent bronchial
vasodilator (4), and may also mediate cytotoxic effects on airway
epithelia (24). In the present study, we have used mice rendered
deficient for NOS2 production in a model of allergic airway disease to
more clearly define the activity of this molecule in disease
pathogenesis.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The NOS2-deficient mice and wild-type controls used in these studies were derived as described elsewhere (25). Cells from the NOS2 mutants were deficient for NO production in response to inductive regimens, which included LPS and/or IFNs. The mice were bred in the Animal Breeding Establishment of the Australian National University and housed in an approved containment facility. Mice were treated according to Australian National University Animal Welfare guidelines, and age- and sex-matched animals were used in these studies.
Sensitization and aeroallergen treatment of mice
A murine model of allergic airways disease was established as
previously described (26). In this model, exposure to aeroallergen
induces airway inflammation and AHR, which are closely associated with
eosinophilia and localized expression of Th2-type cytokines,
particularly IL-4 and IL-5. Briefly, mice (8–10 wk of age) were
sensitized by two i.p. injections with 50 µg OVA (Grade V; Sigma, St.
Louis, MO) in 1 mg alhydrogel (Commonwealth Serum Laboratories,
Parkville, Australia) given on day 0 and 12. Nonsensitized controls
received 1 mg of alhydrogel in normal saline. On day 24, sensitized
mice were exposed to aerosolized OVA (10 mg/ml in normal saline) for
three 30-min periods at intervals of 30 min, and the process was
repeated every second day for 6 days. Nonsensitized mice received
aerosolized saline only. The aerosol was generated at 6 L/min by a
nebulizer producing particles of 
3.9 µm diameter into a closed
chamber of 800 cm3. Mice were sacrificed on day 31, 24
h after the last aerosol. In some experiments, groups of mice were
given an i.p. dose of 0.8 mg/mouse of either mAb against murine IFN-
(R4-6A2, American Type Culture Collection, Manassas, VA) or mAb GL113
(a kind gift of Dr. J. Abrams, DNAX Research Institute of Molecular and
Cellular Biology, Palo Alto, CA) as a rat IgG1 isotype control one day
before each course of aeroallergen treatment (i.e., on day 23, 25, 27,
and 29). Sections were cut from paraffin-embedded lung tissues and
stained with May-Grunwald-Giemsa solution for histological studies.
Assessment of bone marrow, blood and lung inflammatory cell populations
Twenty-four h after the final treatment with aeroallergen on the
day of sacrifice (day 31), bronchoalveolar lavage (BAL) was performed
by tracheal cannulation and instillation of 1 ml of normal saline into
the lungs. Each animal was lavaged twice, with 0.8 ml of fluid being
recovered per wash. BAL fluid (BALF) was centrifuged (1500 x
g at 4°C for 5 min), total cell counts were made using
trypan blue, and 106 cells were taken for cytospin (Shandon
Scientific, Cheshire, U.K.). Blood smears were made from samples drawn
from the tail vein immediately before each course of aeroallergen
treatment and on the day of sacrifice. Numbers of eosinophilic cells in
bone marrow were assessed using aspirates collected before aeroallergen
treatment (day 23) and on the day of sacrifice. Cytospin smears of
cells from BALF, blood, and bone marrow were stained with
May-Grunwald-Giemsa solution, and 200 leukocytes were counted on each
slide.
Measurement of eosinophil hematopoiesis in bone marrow
A modification of the method of Stanley was used to assess eosinophil colony formation from bone marrow progenitor cells in vitro (27). Briefly, 24 h after the final treatment with aeroallergen, bone marrow cells were collected and prepared in 1x HBSS containing 15 mM HEPES, 2% FCS, and 50 µg/ml gentamicin. Cells from mice within each group were pooled and cultured at 4 x 105 cells/well in 12-well Luc migration plates (Bacto Laboratories, New South Wales, Australia) in a volume of 400 µl of 0.33% agar (Unipath, Basingstoke, U.K.) in RPMI 1640 medium (JRH Biosciences, Lenexa, KS) containing 10% FCS, 4 mM glutamine, 60 µg/ml gentamicin, 500 U/ml IL-3, and 250 U/ml IL-5 (the cytokines are a kind gift of Dr Andrew Hapel, John Curtin School of Medical Research). Cultures were incubated at 37°C in a humidified atmosphere of 5% CO2 in air for 7 days. Semisolid cultures were then lifted onto microscope slides, air-dried, and stained with May-Grunwald-Giemsa solution. Numbers of colonies containing eosinophils were scored under light microscopy using morphological criteria.
Measurement of AHR
Mice were anesthetized with a mixture of ketamine (Troy Laboratories, Smithfield, Australia) at 60 mg/kg body mass and Rompun (Bayer Australia, Pymble, Australia) at 8 mg/kg given by i.p. injection, and their tracheae were cannulated and connected to a bronchospasm transducer (Ugo Basil 7020) coupled to a Lab Mac/8 computer analysis station (AdInstruments, Sydney, Australia). Analyses were performed during cumulative i.v. administration of the spasmogen ß-methacholine as detailed elsewhere (26). The increase in respiratory overflow volume provoked by ß-methacholine is represented as a percentage of maximal overflow volume (100%) obtained by totally occluding the tracheal cannula.
Determination of OVA-specific Igs
Two hours before the first aeroallergen treatment and on the day of sacrifice, blood samples were taken for determination of serum anti-OVA IgG1, IgG2a, and IgE Ab titers by standard ELISA. Biotinylated goat anti-mouse IgG1 or IgG2a (Biosource International, Camarillo, CA) or rat anti-mouse IgE (PharMingen, San Diego, CA) were used, and each plate contained a previously screened isotype standard.
Preparation of CD4+ T cell populations from lung and spleen
Mice were anesthetized with ketamine and Rompun as above and their lungs perfused with 1 ml of collagenase (0.1% in PBS) injected through the trachea. Tracheae were tied with surgical sutures and the lungs removed immediately and incubated at 37 C in HBSS for 60 min. Spleens were also collected into HBSS. Organs were then gently homogenized, filtered through sterile surgical gauze, RBC were lysed by osmotic shock, and the preparations were centrifuged (1500 x g for 10 min) and resuspended in DMEM supplemented with 10% FCS, 50 µM 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, and antibiotics (complete medium). The cell preparations were labeled with L3T4 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and passed through MiniMACS Separation Columns (Miltenyi Biotec). Eluates were shown by flow cytometry to comprise >95% CD4+ T cells.
Determination of cytokine production
Cytokine levels were assayed in BALF taken on the day of
sacrifice and in supernatants overlying cultures of CD4+ T
cells from lung and spleen. Purified CD4+ T cell
populations were cultured at 5 x 105 cells/well in
complete medium on anti-CD3 mAb (2C11, 50 µg/ml)-coated 96-well
microtiter plates (Nunc, Roskilde, Denmark) at 37°C for 72 h.
Supernatants were then harvested for determination of cytokine
production by standard ELISA. The Ab pairs used for cytokine detection
were as follows: IL-4, 11B11 (American Type Culture
Collection) and BVD6-24G2 (PharMingen); IL-5, TRFK-5 (28) and
TRFK-4 (PharMingen); IFN-, R4-6A2 (American Type Culture
Collection), and a polyclonal rabbit anti-mouse IFN- Ab made in
our laboratory; TNF-, TN3-19.12 (a kind gift of Dr. R. Schreiber,
Washington University, St. Louis, MO), and a polyclonal rabbit
anti-mouse TNF Ab (a kind gift of Mrs. S. Fordham, John Curtin
School of Medical Research); IL-10, SXC-1, and SXC-2 (kind gifts of Dr.
K. Moore, DNAX) (29); and IL-12, C17.8, and C15.6 (kind gifts of Dr. G.
Trinchieri, Wistar Institute, Philadelphia, PA).
Measurement of reactive nitrogen intermediates (RNI)
Sera were collected 24 h after the final treatment with aeroallergen and tested for the presence of RNI (nitrites or nitrates) according to established methods (30). Briefly, nitrite was measured by addition of 100 µl of Griess reagent to 30 µl of test sample. Protein was removed by the addition of 100 µl of trichloroacetic acid followed by centrifugation, and the optical density of each sample was read at 540 nm with a reference at 650 nm using a microplate reader. For nitrate measurements, the nitrate was first converted to nitrite by incubation with nitrate reductase and NADPH (Boehringer Mannheim, Mannheim, Germany) for 20 min. Results were quantified by reading against appropriate standard curves.
Statistical analyses
Data were expressed as mean ± SEM, and differences between experimental groups within each experiment were analyzed using the unpaired Student’s t test.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Levels of RNI were measured in sera to confirm that NOS2
gene-deficiency resulted in reduced levels of NO induced during the
course of allergic inflammation. The concentrations of nitrates in sera
from OVA-treated wild-type (NOS2+/+) mice were
significantly greater than those from control (saline-treated)
wild-type or NOS2-deficient mice (Fig. 1
). In contrast, RNI were detected in
OVA-treated NOS2-deficient mice only at backgound levels (Fig. 1).
These results indicate that increased production of NO is not induced
during allergic lung inflammation in the mutant mice.
|
Aerosol challenge of wild-type mice sensitized with OVA normally
leads to widespread eosinophilic inflammation, primarily in the
peribronchial and perivascular regions as we have previously reported
(26). Therefore, we initially examined blood smears taken from
aerosol-treated wild-type mice and found markedly elevated numbers of
eosinophils in the bloodstream of those given OVA (Fig. 2). A significant blood eosinophilia was
first detected on day 28, after the second day of exposure to
aeroallergen, with peak counts recorded on day 31, after the full
course of treatment. Although blood eosinophil numbers had also
increased in OVA-treated NOS2-/- mice by day 28, they
remained relatively constant thereafter until sacrifice and were
significantly lower than the rising counts recorded in wild-type mice
after both the third (p < 0.05) and fourth
(p < 0.01) aeroallergen treatments.
|
).
Eosinophils, whilst rarely detected in saline-treated wild-type or
NOS2-/- mice, were the predominant leukocyte type in BALF
from aeroallergen-treated mice. However, their numbers in
NOS2-/- mice were consistently less than half of those
found in wild-type animals. Other leukocyte populations, particularly
lymphocytes and macrophages, were present in BALF in much smaller
numbers, although these were significantly elevated in the NOS2 mutant
mice. In the case of lymphocytes, a 20-fold increase over
saline-treated mutants was recorded compared with a 5-fold increase in
the wild-type mice, indicating that these noneosinophilic components of
the allergic inflammatory response are somewhat enhanced in the
NOS2-deficient animals.
|
Eosinophil development and efflux is not suppressed in NOS2-deficient mice
We next studied the development of eosinophils in the bone marrow
and their efflux in NOS2-deficient mice in light of the marked
decreases in numbers of these cells in their blood and airways
following aeroallergen treatment. Analyses of bone marrow cells taken
from OVA-treated NOS2-/- and wild-type mice in
colony-forming assays revealed no significant differences in the
percentages of colonies containing eosinophils after 7 days in culture
(Table II). In addition, similar
decreases in numbers of eosinophilic cells were found in the bone
marrow of sensitized NOS2 mutants and wild-type mice after aerosol
treatment with OVA (Table III). These
findings indicate that the reduction in circulatory and pulmonary
eosinophil numbers observed in NOS2-deficient mice are due neither to
suppression of their maturation nor of their efflux from the bone
marrow in response to aeroallergen challenge.
|
|
In our model, airway eosinophilic inflammation following
aeroallergen challenge is directly associated with AHR to the
spasmogen, ß-methacholine (26). Our findings of markedly decreased
eosinophilic inflammation and airway disease in sensitized
NOS2-deficient mice treated with aerosolized OVA prompted us to study
the development of AHR in these animals. As shown in Fig. 3, AHR in allergic wild-type mice
increased with dosage of ß-methacholine to a maximum of 60% airway
occlusion over the baseline levels seen in controls. Similar high
levels of AHR were found in the OVA-treated NOS2 mutants, although
these were recorded over baseline levels that were markedly higher than
those found in the wild-type mice. These elevated basal readings may
reflect removal of the known bronchodilatory influence of inducible NO
in the mutants. Nevertheless, it appeared that the diminished airway
allergic inflammatory infiltrate following OVA treatment of these mice
was still sufficient to provoke substantial AHR.
|
production does not greatly modify the type 2
cytokine-driven immune response in allergic NOS2-/- mice
We next compared the cytokine profiles of CD4+ T cells
and specific Ab responses in aeroallergen-treated NOS2-/-
and wild-type mice. Given the correlation between allergic airway
disease and expression of type 2 cytokines by lung T cells, together
with the proposition that local NO production promotes airway
inflammation through down-regulation of T cell-derived IFN-, it was
of interest to determine whether the marked decrease in blood and
airway eosinophilia seen in the aeroallergen-treated NOS2 mutants might
reflect a shift in the characteristics of the immune response in these
animals. As shown in Fig. 4, activated
lung and spleen CD4+ T cells from control, nonallergic
NOS2-/- and wild-type mice secreted similar levels of
IFN-; however, neither IL-4 nor IL-5 was detected. In contrast,
while CD4+ T cells from OVA-treated wild-type animals
produced significantly less IFN- than those from saline-treated
controls, there was pronounced expression of both IL-4 and IL-5,
particularly among cells from the lungs. Similar patterns of type 2
cytokine expression were found in cells from OVA-treated NOS2 mutants;
however, both lung and splenic T cells from these animals also secreted
very high levels of IFN-. In addition, IL-12, TNF-, and IL-10
were detected at similar levels only marginally above background in
BALF from both wild-type and mutant mice treated with OVA (data not
shown), contrasting with the elevated levels of IL-5 (55 U/ml in
wild-type; 48 U/ml in NOS2 mutants) found in these fluids.
|
). While significantly lower
levels of OVA-specific IgE were found in sensitized NOS2 mutants
compared with wild-type mice, titers were also similar after
aerosolization. However, NOS2 deficiency increased levels of specific
IgG2a, albeit at comparatively low titers, particularly after OVA
aerosol challenge.
|
production and in levels of serum IgG2a against the allergen. However,
neither the characteristic type 2 cytokine-driven specific IgG1 and IgE
responses nor the production of IL-4 and IL-5 by lung T cells were
modified in allergic NOS2-/- mice.
Depletion of IFN- reverses inhibition of airway inflammation in
NOS2-/- mice
These pronounced increases in IFN- production might account for
the decreased eosinophilic inflammation and, therefore, airway disease,
seen in aeroallergen-treated NOS2-deficient mice. To test this
hypothesis, we treated the mutants with mAbs against IFN- during
their course of treatment with OVA. As shown in Fig. 6, the inhibition of blood eosinophilia
from day 28 (after the second treatment with aerosolized OVA) recorded
in NOS2-/- mice given control mAb was abolished in those
given mAb against IFN-. Similarly, the marked reduction in
eosinophil numbers in BALF seen in allergic mutants was reversed in
mice depleted of this factor (Fig. 7). In
addition, the symptoms of severe disease as described above in
wild-type mice were evident in the mAb-treated mutants (data not
shown). Neutralization of IFN- activity did not significantly affect
levels of AHR in the mutant mice (data not shown). Thus, it appears
that IFN- was responsible for the suppression of eosinophilic
inflammation and allergic pathology in NOS2-/- mice
despite their capacity for continued production of IL-5.
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
upon restimulation in vitro, but not by any obvious
change in local production of IL-4, IL-5, IL-10, IL-12, or TNF-, nor
by major changes in the largely IgG1 and IgE OVA-specific serum Ig
profiles in the mutants. The markedly enhanced production of IFN- in
the NOS2-/- mice was apparently responsible for the
suppression of both eosinophilia and disease, as in vivo depletion of
this factor restored allergic inflammation and pathology in these
animals.
Our findings are in partial agreement with those of Feder and
colleagues, who recently showed that chemical inhibition of NO activity
suppressed pulmonary eosinophilic inflammation in a murine model of
allergic airway disease (17). However, these authors attributed the
reduction in inflammation to NO catalyzed by enzymes other than NOS2
and speculated that localized NO production enhances extravasation of
circulating eosinophils into the lung. In contrast, our study, using
mice with a specific mutation, has clearly shown that NOS2 deficiency
leads to a IFN--mediated suppression of both pulmonary and
circulating eosinophilia and allergic airways disease following
aeroallergen challenge.
The severity of clinical asthma appears to correlate with the degree of
airway inflammation and, therefore, the level of local Th2 cell
activity (31). In murine models of allergic airway inflammation,
aeroallergen challenge of sensitized mice normally leads to widespread
eosinophilia, which correlates directly with disease (26, 32, 33, 34). In
addition, we have shown that eosinophilia is, in turn, critically
dependent on IL-5 in vivo (26) and that type 1 cytokine-mediated
suppression of eosinophilia and disease is accompanied by a marked
down-regulation of local IL-5 production (35). Therefore, it was
somewhat surprising to find that T cell isolates from both the lungs
and spleens of aeroallergen-treated NOS2-/- mutants
retained the capacity to secrete high levels of both IL-4 and IL-5, as
in wild-type animals, in the face of their ability to produce large
amounts of IFN- upon restimulation in vitro, particularly given the
established role of IFN- as a potent suppressor of Th2 cell activity
(35, 36, 37). These findings lead us to suggest that the suppressive
effects of IFN- may be countered by the generation of strong Th2
responses with IL-4 and IL-5 secretion, such as in our model, and that
high local levels of IL-4 promote Th2 cell development despite
significant local IFN- production. In this context, we and others
have recently reported that local virus infections that normally induce
IFN- production did not suppress eosinophilic airway inflammation in
similar models of allergic airway disease (35, 38).
The enhanced T cell production of IFN- observed in the
allergen-treated NOS2 mutants is in accordance with the finding that
treatment of mice with inhibitors of NO production leads to marked
increases in IFN- levels during staphylococcal infection (39).
Similar enhancement of IFN- production has recently been reported in
studies using NOS2-/- mutants infected with the
protozoan, Leishmania major (40, 41), Mycobacterium
tuberculosis (42), or viruses, including ectromelia virus and
influenza virus, contributing to rapid viral clearance in the latter
(G.K., unpublished observations). How NO influences IFN- gene
expression is unclear at this stage. However, recent evidence indicates
that NO may regulate the activity of NF-
B transcription factors and
protein tyrosine kinases belonging to the src kinase,
mitogen-activated protein (MAP) kinase, and Janus kinase (JAK) families
(43, 44, 45, 46), the latter with major implications for IFN- signaling
(47). It has also been suggested that NO may down-regulate Th1-type
immune responses and, in this way, diminish IFN- production (40).
The delineation of mechanisms that regulate the development of
different T cell subsets is of obvious importance for increased
understanding of the pathogenesis of a number of diseases including
allergy. In this regard, recent in vitro studies have indicated that NO
may act differentially on activated murine and human Th cell
populations by inhibiting Th1 cytokine production but promoting the
expression of Th2-type cytokines (48, 49), although these data remain
controversial (50). Nevertheless, it has been proposed that inducible
NO, produced in the asthmatic airways by epithelial cells, macrophages,
and possibly Th1-type cells, has an inhibitory effect on Th1-type cells
and their expression of IFN-, facilitating the activation of
Th2-type cells and the development of allergic inflammation (21). The
greatly increased local expression of IFN- we recorded in
allergen-treated NOS2-/- mutants lends partial support to
this hypothesis. However, it was clear from our data that the
IFN--mediated protection from airway inflammation in these animals
was not due to suppression of T cell production of IL-4 and IL-5 by
this factor.
Our findings in NOS2-/- mutant mice indicate that
inhibitors of NOS2 may be effective in suppressing allergic
inflammation, particularly in the airways. They also suggest that the
effects of such compounds would largely be mediated through IFN-
activity in the face of continued production of Th2-type cytokines.
These data suggest several potential mechanisms whereby allergic
inflammation may be suppressed. The inhibition of eosinophilia recorded
not only in BALF but also in the circulation of aeroallergen-treated
NOS2 mutants and its reversal by mAbs directed against IFN- raised
the possibility that this factor may down-regulate inflammatory cell
hematopoiesis, either directly or via intermediate molecules, possibly
including NOS2 itself. However, our studies using bone marrow cells
indicate that eosinophil development and efflux from the bone marrow
following aeroallergen challenge is unaffected by NOS2 deficiency and
suggest that the inhibitory effects of IFN- may occur at a later
stage in eosinophilic inflammation, such as the subsequent maturation
of these cells in the circulation. In addition, IFN- may directly
influence the activity of mature inflammatory cells and, in this
respect, has been shown to inhibit degranulation and release of
inflammatory mediators from both eosinophils (51) and mast cells (52).
In the latter case at least, this suppression may be mediated by
IFN--induced NO (53). Selective inhibition of NO production might
also protect against allergic pulmonary inflammation independently of
increased IFN- activity, our present data notwithstanding. It has
recently been shown that NO specifically disrupts Fas receptor-mediated
apoptosis of eosinophils (54), while its vasodilatory properties (4)
and chemotactic activity for a variety of cell types, including
eosinophils (55), may enhance their recruitment into allergic airways
(17).
The lack of obvious differences between AHR measured in aeroallergen-treated wild-type and NOS2-/- mice, despite suppressed pulmonary eosinophilic inflammation in the latter, probably reflects the continued production of levels of eosinophil inflammatory mediators sufficient to provoke such hyperreactivity. Our previous work in this model has clearly demonstrated a close association between local eosinophilic inflammation and AHR (26). However, it should be noted that the elevated AHR readings in OVA-challenged mice were recorded over baseline levels in control (saline-treated) animals that were significantly greater in the NOS2 mutants. Deficient NO production in these mice may have been responsible for this spontaneous AHR, given the reported, albeit variable, bronchodilatory activity of this molecule (22, 23).
In conclusion, we have shown that eosinophilic inflammation and related
manifestations of allergic airway disease are markedly suppressed in
mice deficient for NOS2 production, apparently due to pronounced
IFN- expression in these animals. Our results support the hypothesis
that NO promotes allergic inflammation and indicate that specific
inhibitors of NOS2 may be worthwhile components of therapeutic
strategies against allergic diseases, including asthma.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to: Dr. Alistair J. Ramsay, Division of Immunology and Cell Biology, John Curtin School of Medical Research, Australian National University, Canberra, Australia. E-mail address:
3 Abbreviations used in this paper: NOS2, nitric oxide synthase 2; NO, nitric oxide; AHR, airway hyperreactivity; BALF, bronchoalveolar lavage fluid; RNI, reactive nitrogen intermediates; MAP kinase, mitogen-activated protein kinase; JAK, Janus kinase.
Received for publication March 30, 1998. Accepted for publication September 8, 1998.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene expression in steroid-resistant asthma. J. Exp. Med. 181:33.-deficient mice infected with Leishmania major. J. Exp. Med. 179:1367., IL-10 and inducible nitric oxide synthase in human T cells by cyclic AMP-dependent signal transduction pathway. Immunology 91:361.[Medline]
inhibits serotonin release from mouse peritoneal mast cells. Eur. J. Immunol. 21:2559.[Medline]
-induced suppression of exocytosis. J. Immunol. 159:1444.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  A. S. Naura, R. Datta, C. P. Hans, M. Zerfaoui, B. M. Rezk, Y. Errami, M. Oumouna, K. Matrougui, and A. H. Boulares Reciprocal regulation of iNOS and PARP-1 during allergen-induced eosinophilia Eur. Respir. J., February 1, 2009; 33(2): 252 - 262. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. G. Martin and T. Jo Genetic Differences in Airway Smooth Muscle Function Proceedings of the ATS, January 1, 2008; 5(1): 73 - 79. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Takemoto, K. Ogino, M. Shibamori, T. Gondo, Y. Hitomi, T. Takigawa, D.-H. Wang, J. Takaki, H. Ichimura, Y. Fujikura, et al. Transiently, paralleled upregulation of arginase and nitric oxide synthase and the effect of both enzymes on the pathology of asthma Am J Physiol Lung Cell Mol Physiol, December 1, 2007; 293(6): L1419 - L1426. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Brindicci, K. Ito, P. J. Barnes, and S. A. Kharitonov Effect of an Inducible Nitric Oxide Synthase Inhibitor on Differential Flow-Exhaled Nitric Oxide in Asthmatic Patients and Healthy Volunteers Chest, August 1, 2007; 132(2): 581 - 588. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Batra, T. Pratap Singh, U. Mabalirajan, A. Sinha, R. Prasad, and B. Ghosh Association of inducible nitric oxide synthase with asthma severity, total serum immunoglobulin E and blood eosinophil levels Thorax, January 1, 2007; 62(1): 16 - 22. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ferlito, K. Irani, N. Faraday, and C. J. Lowenstein Nitric oxide inhibits exocytosis of cytolytic granules from lymphokine-activated killer cells PNAS, August 1, 2006; 103(31): 11689 - 11694. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Bhandari, R. Choo-Wing, S. P. Chapoval, C. G. Lee, C. Tang, Y. K. Kim, B. Ma, P. Baluk, M. I. Lin, D. M. McDonald, et al. Essential role of nitric oxide in VEGF-induced, asthma-like angiogenic, inflammatory, mucus, and physiologic responses in the lung PNAS, July 18, 2006; 103(29): 11021 - 11026. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A.-K. Larsson, M. Back, J. Hjoberg, and S.-E. Dahlen Inhibition of Nitric-Oxide Synthase Enhances Antigen-Induced Contractions and Increases Release of Cysteinyl-Leukotrienes in Guinea Pig Lung Parenchyma: Nitric Oxide as a Protective Factor J. Pharmacol. Exp. Ther., October 1, 2005; 315(1): 458 - 465. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. G. Que, L. Liu, Y. Yan, G. S. Whitehead, S. H. Gavett, D. A. Schwartz, and J. S. Stamler Protection from Experimental Asthma by an Endogenous Bronchodilator Science, June 10, 2005; 308(5728): 1618 - 1621. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Peters-Golden The Alveolar Macrophage: The Forgotten Cell in Asthma Am. J. Respir. Cell Mol. Biol., July 1, 2004; 31(1): 3 - 7. [Full Text] [PDF]
|
|
|
|
 |
|
 J. Hjoberg, S. Shore, L. Kobzik, S. Okinaga, A. Hallock, J. Vallone, V. Subramaniam, G. T. De Sanctis, J. A. Elias, J. M. Drazen, et al. Expression of nitric oxide synthase-2 in the lungs decreases airway resistance and responsiveness J Appl Physiol, July 1, 2004; 97(1): 249 - 259. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. L. M. Ricciardolo, P. J. Sterk, B. Gaston, and G. Folkerts Nitric Oxide in Health and Disease of the Respiratory System Physiol Rev, July 1, 2004; 84(3): 731 - 765. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Lagente, E. Naline, I. Guenon, M. Corbel, E. Boichot, J.-L. Burgaud, P. Del Soldato, and C. Advenier A Nitric Oxide-Releasing Salbutamol Elicits Potent Relaxant and Anti-Inflammatory Activities J. Pharmacol. Exp. Ther., July 1, 2004; 310(1): 367 - 375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. C. Dorman, R. Sehmi, G. M. Gauvreau, R. M. Watson, R. Foley, G. L. Jones, J. A. Denburg, M. D. Inman, and P. M. O'Byrne Kinetics of Bone Marrow Eosinophilopoiesis and Associated Cytokines after Allergen Inhalation Am. J. Respir. Crit. Care Med., March 1, 2004; 169(5): 565 - 572. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Okamoto, K. Gohil, E. I. Finkelstein, P. Bove, T. Akaike, and A. van der Vliet Multiple contributing roles for NOS2 in LPS-induced acute airway inflammation in mice Am J Physiol Lung Cell Mol Physiol, January 1, 2004; 286(1): L198 - L209. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Rodriguez, A. C. Keller, E. L. Faquim-Mauro, M. S. de Macedo, F. Q. Cunha, J. Lefort, B. B. Vargaftig, and M. Russo Bacterial Lipopolysaccharide Signaling Through Toll-Like Receptor 4 Suppresses Asthma-Like Responses Via Nitric Oxide Synthase 2 Activity J. Immunol., July 15, 2003; 171(2): 1001 - 1008. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. H. Boulares, A. J. Zoltoski, Z. A. Sherif, P. Jolly, D. Massaro, and M. E. Smulson Gene Knockout or Pharmacological Inhibition of Poly(ADP-Ribose) Polymerase-1 Prevents Lung Inflammation in a Murine Model of Asthma Am. J. Respir. Cell Mol. Biol., March 1, 2003; 28(3): 322 - 329. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Birrell, K. McCluskie, E.-B. Haddad, C. H. Battram, S. E. Webber, M. L. Foster, M. H. Yacoub, and M. G. Belvisi Pharmacological Assessment of the Nitric-Oxide Synthase Isoform Involved in Eosinophilic Inflammation in a Rat Model of Sephadex-Induced Airway Inflammation J. Pharmacol. Exp. Ther., March 1, 2003; 304(3): 1285 - 1291. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F L M Ricciardolo Multiple roles of nitric oxide in the airways Thorax, February 1, 2003; 58(2): 175 - 182. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. R. Eynott, N. Paavolainen, D. A. Groneberg, A. Noble, M. Salmon, P. Nath, S.-Y. Leung, and K. F. Chung Role of Nitric Oxide in Chronic Allergen-Induced Airway Cell Proliferation and Inflammation J. Pharmacol. Exp. Ther., January 1, 2003; 304(1): 22 - 29. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. M. W. Janssen-Heininger, R. L. Persinger, S. H. Korn, C. Pantano, B. McElhinney, N. L. Reynaert, R. C. J. Langen, K. Ckless, P. Shrivastava, and M. E. Poynter Reactive Nitrogen Species and Cell Signaling: Implications for Death or Survival of Lung Epithelium Am. J. Respir. Crit. Care Med., December 15, 2002; 166(12): S9 - 16. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. D. Lang, P. J. McArdle, P. J. O'Reilly, and S. Matalon Oxidant-Antioxidant Balance in Acute Lung Injury Chest, December 1, 2002; 122(6_suppl): 314S - 320S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Koarai, M. Ichinose, H. Sugiura, M. Tomaki, M. Watanabe, S. Yamagata, Y. Komaki, K. Shirato, and T. Hattori iNOS depletion completely diminishes reactive nitrogen-species formation after an allergic response Eur. Respir. J., September 1, 2002; 20(3): 609 - 616. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Q. Truong-Tran, R. E. Ruffin, P. S. Foster, A. M. Koskinen, P. Coyle, J. C. Philcox, A. M Rofe, and P. D. Zalewski Altered Zinc Homeostasis and Caspase-3 Activity in Murine Allergic Airway Inflammation Am. J. Respir. Cell Mol. Biol., September 1, 2002; 27(3): 286 - 296. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Careau, J. Sirois, and E. Y. Bissonnette Characterization of Lung Hyperresponsiveness, Inflammation, and Alveolar Macrophage Mediator Production in Allergy Resistant and Susceptible Rats Am. J. Respir. Cell Mol. Biol., May 1, 2002; 26(5): 579 - 586. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. R. Pitt and C. M. St. Croix Complex Regulation of iNOS in Lung Am. J. Respir. Cell Mol. Biol., January 1, 2002; 26(1): 6 - 9. [Full Text] [PDF]
|
|
|
|
|
|
C. E. Cross, A. van der Vliet, and J. P. Eiserich Peroxidases Wheezing Their Way into Asthma Am. J. Respir. Crit. Care Med., October 1, 2001; 164(7): 1102 - 1103. [Full Text] [PDF]
|
|
|
|
|
|
A. DUGUET, H. IIJIMA, S.-Y. EUM, Q. HAMID, and D. H. EIDELMAN Eosinophil Peroxidase Mediates Protein Nitration in Allergic Airway Inflammation in Mice Am. J. Respir. Crit. Care Med., October 1, 2001; 164(7): 1119 - 1126. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. IIJIMA, A. DUGUET, S.-Y. EUM, Q. HAMID, and D. H. EIDELMAN Nitric Oxide and Protein Nitration are Eosinophil Dependent in Allergen-Challenged Mice Am. J. Respir. Crit. Care Med., April 1, 2001; 163(5): 1233 - 1240. [Abstract] [Full Text]
|
|
|
|
|
|
G. R. Strohmeier, J. H. Walsh, E. S. Klings, H. W. Farber, W. W. Cruikshank, D. M. Center, and M. J. Fenton Lipopolysaccharide Binding Protein Potentiates Airway Reactivity in a Murine Model of Allergic Asthma J. Immunol., February 1, 2001; 166(3): 2063 - 2070. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. STEUDEL, M. KIRMSE, J. WEIMANN, R. ULLRICH, J. HROMI, and W. M. ZAPOL Exhaled Nitric Oxide Production by Nitric Oxide Synthase-deficient Mice Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): 1262 - 1267. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Trifilieff, Y. Fujitani, F. Mentz, B. Dugas, M. Fuentes, and C. Bertrand Inducible Nitric Oxide Synthase Inhibitors Suppress Airway Inflammation in Mice Through Down-Regulation of Chemokine Expression J. Immunol., August 1, 2000; 165(3): 1526 - 1533. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. P. Sanders Nitric Oxide in Asthma . Pathogenic, Therapeutic, or Diagnostic? Am. J. Respir. Cell Mol. Biol., August 1, 1999; 21(2): 147 - 149. [Full Text]
|
|
|
|
 |
|
 G. T. De Sanctis, J. A. MacLean, K. Hamada, S. Mehta, J. A. Scott, A. Jiao, C. N. Yandava, L. Kobzik, W. W. Wolyniec, A. J. Fabian, et al. Contribution of Nitric Oxide Synthases 1, 2, and 3 to Airway Hyperresponsiveness and Inflammation in a Murine Model of Asthma J. Exp. Med., May 17, 1999; 189(10): 1621 - 1630. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
