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*
Novartis Horsham Research Centre, Horsham, United Kingdom;
Immuno-Hematology Group, Hôpital La Pitié Salpêtrière, Paris, France; and
Roche Bioscience, Palo Alto, CA
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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). In vitro exposure of primary cultures of murine
lung fibroblasts to a NO donor, hydroxylamine, induced a dose-dependent
release of macrophage inflammatory protein-2 and monocyte
chemoattractant protein-1. Our results suggest that lung inflammation
after allergen challenge in mice is partially dependent on NO produced
mainly by iNOS. NO appears to increase lung chemokine expression and,
thereby, to facilitate influx of inflammatory cells into the
airways. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The pathophysiological role of NO in the airways is unclear, but endogenous NO has been shown to have a beneficial role such as neuromediation of the bronchodilator innervation in human tracheal segments in vitro (2) and in guinea pigs in vivo (3). Furthermore, NO may also be an important mediator of mucociliary clearance by increasing ciliary beat frequency in bovine epithelial cells (4). On the other hand, NO could also have deleterious effects such as increase in pulmonary blood flow (5), plasma exudation (6), and mucus hypersecretion (7).
In the airways, at least one or more isoforms of NOS were shown to be expressed in endothelial or epithelial cells, macrophages, mast cells, and both vascular and bronchial smooth muscle cells (8). Due to the lack of pharmacological tools, it is unclear which cell type(s) or isoform(s) produce NO in the lung. However, there is evidence for the expression of iNOS in the epithelium of asthmatic lungs but not in those of normal subjects (9). Similarly, both human and murine epithelial cells in culture show an increased expression of iNOS and production of NO after exposure to inflammatory cytokines (10, 11). Recently an increase in exhaled NO has been described in asthmatic patients when compared with healthy volunteers (12, 13, 14, 15), which could be reduced by pretreatment with aminoguanidine, an iNOS inhibitor, suggesting a role of this NOS isoform in the airway pathophysiology (16).
It has been postulated by virtue of its ability to inhibit the
expansion of Th1 cells and their production of IFN- that NO can
facilitate the expansion of Th2 cells, which are key cells for the
development of inflammation in asthma (17). Moreover, NO
has also been shown to be a survival factor for human blood eosinophils
in vitro (18). To study the possible involvement of NO in
the maintenance of the inflammatory reaction occurring in the airways
in vivo, we studied the effects of NOS inhibitor treatment in a murine
allergen-driven model of lung inflammation developed in our group
(19). Both selective iNOS inhibitors,
S-ethylisothiourea (EIT) and
2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT)
(20, 21), and a nonselective iNOS inhibitor,
NG-nitro-L-arginine
methyl ester (L-NAME), were used in this
study.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice (25–30 g) were immunized i.p. with 10 µg of OVA, (grade V; Sigma, St. Louis, MO) in 0.2 ml of alum (Serva, Heidelberg, Germany) on days 0 and 14. On day 21, animals were challenged for 20 min with a nebulized solution of OVA in PBS (50 mg/ml) or with PBS alone. At the specified time point after the challenge, mice were anaesthetized with an i.p. injection of urethane. The NOS inhibitors were dissolved in PBS (pH 7.4). Drug or PBS as a control were given twice a day by i.p. injection in 0.1 ml (22). In the first set of experiments, treatment was applied from day 14 to day 20, and it was applied from day 20 to the day of sacrifice in the second set.
Determination of enzymatic NOS activity
Incorporation of 14C
L-arginine was evaluated on lung extract from mice
sacrificed 3 days after the challenge as described elsewhere
(23). Briefly, lung preparations were digested after four
cycles of freezing on dry ice in a buffer containing 5 mM dithiotreitol
and the following protease inhibitors: 1 µg/ml leupeptin, 1 µg/ml
pepstatin, 40 µg/ml bestatin, 10 µg/ml chymostatin, 50 µg/ml
N
-p-tosyl-L-lysine-chloromethyl
ketone, and 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride (all from
Sigma). They were then centrifuged at 20,000 x g for
1 h at 4°C. Twenty microliters of the supernatant was then
incubated for 15 min at 37°C with 50 mM
K2PO4, 1 mM
MgCl2, 0.2 mM CaCl2, 50 mM
L-valine, 1 mM
L-citrulline, 0.3 mM NADPH, 0.66 µCi/ml
14C-L-arginine, and 20 µM
cold L-arginine. The reaction was stopped with
the resin suspension, and after vigorous mixing the supernatants were
transferred into scintillation vials. The reaction was performed with
or without EGTA (2 mM) to determine the calcium-independent activity of
the iNOS. An inhibitor of NOS enzymes (L-NAME, 2
mM) was also used in each condition to determine the background
count.
Determination of total serum IgE level
After the anesthesia on day 24, blood was taken from the abdominal aorta. Serum was prepared and IgE Ab titers were determined by ELISA as previously described (24).
Assessment of lung and peripheral blood inflammatory cell
At specified time points the trachea was cannulated and bronchoalveolar lavage (BAL) was performed by injecting 0.3 ml of PBS four times into the lung via the trachea. The fluid was then immediately withdrawn, and the cell suspension was stored on ice. Blood samples were taken from the tail. Total cell count was measured for both BAL and blood. Cytospin preparation (Shandon Scientific, Cheshire, U.K.) and blood smears were prepared for BAL and blood samples, respectively. Cells were stained with Diff-Quik (Baxter Dade, Dudingen, Switzerland), and a differential count of 200 cells was performed using standard morphological criteria.
Purification of lung T cells and determination of cytokine production
On day 24, after four repeated lavages for assessment of the
inflammatory cells, the lungs were perfused via the right ventricle
with 5 ml of PBS containing 100 U/ml of heparin to remove blood and
intravascular leukocytes. The lungs were then removed, cleaned of
extraneous tissues, and placed into ice-cold DMEM containing 10% FCS,
50 µM 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM
HEPES, and 50 µg/ml gentamicin. The lungs were gently homogenized and
filtered through a 70-µm filter, and the lymphocytes were purified
over a single-step Ficoll gradient. The lymphocyte fraction was then
incubated with a mixture of rat Ab against CD45R/B220 and CD24
(PharMingen, San Diego, CA). B cells and NK cells were then eliminated
by adding magnetic beads coated with anti-rat IgG (Dynal, Oslo,
Norway). Purified T cells were plated at a concentration of 1 x
105 cells/well on anti-CD3-coated microtiter
plates (2C11; 50 µg/ml) in the presence of human rIL-2 (200 U/ml).
After 4 days, the supernatant was harvested, and cytokine production
(IL-4, IL-5, and IFN-) was determined by ELISA as described
previously (19). The limits of detection were as follows:
IL-4, 0.5 U/ml; IL-5, 100 U/ml; and IFN-, 50 U/ml.
Purification and survival of eosinophils in vitro
Instead of a single OVA challenge, immunized mice were challenged daily from day 21 to day 25 and were sacrificed at day 28. This protocol, based on our previous study (25), induces a selective eosinophil infiltration with less than 2% neutrophils or lymphocytes, thus allowing eosinophils to be purified. After anesthesia, BAL was performed by 10 repeated lavages with 0.5 ml of ice-cold DMEM containing 5% FCS, 50 µM 2-ME, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, and 50 µg/ml gentamicin. The cells were washed and macrophages removed from the cell suspension by adherence to plastic and by centrifugation at 100 x g for 20 min. Eosinophil purity in the supernatant was determined by staining cytospin preparations with Diff-Quik. In all of the experiments, eosinophil purity was between 94 and 98%.
Survival of eosinophils was evaluated based on a previous study (18). After purification, eosinophils (2 x 105 cells/well) were cultured for 48 h in 96-well plates in either medium alone or in medium supplemented with 300 µM hydroxylamine (Sigma). Eosinophil viability was determined with the MTT viability kit (Boehringer Mannheim, Rotkreuz, Switzerland). Ten microliters of MTT (5 mg/ml in PBS) was added to each well and incubated for 3 h before addition of 100 µl of solubilization buffer (10% SDS in 0.01 M HCl). Plates were then incubated overnight, and absorbance was measured at 570–660 nm.
Histology and immunostaining
Four to six mice per group were used for histological observations. At the specified time point after the aerosol exposure, the lungs were inflated through the trachea with 4% formaldehyde solution in PBS (pH 7.4) under a constant pressure of 150 mm of water. After 2 h, the lungs were removed from the thoracic cavity, cleared of extraneous tissue, and immersed in 4% formaldehyde for 1 h. Lungs were then routinely embedded in paraffin, and 4-µm sections were cut and mounted on glass slides precoated with poly-L-lysine (Sigma). Sections were deparaffinized for 20 min in xylene, dehydrated for 10 min in 100% ethanol, and then washed with PBS for 10 min. Before iNOS staining, endogenous peroxidase activity was inhibited with 0.3% H2O2 in PBS for 15 min. Slides were incubated overnight at 4°C with an anti-iNOS Ab (1/500; clone (M-19)-G; Santa Cruz Biotechnology, Santa Cruz, CA) or with an isotype-matched control Ab (rabbit IgG; 1/500; Sigma) and were revealed using an immunoperoxidase kit (Vector Laboratories, Burlingame, CA).
Preliminary experiments had shown the peak of eosinophilic infiltration in the parenchyma to occur 2 days after the challenge. Therefore, to study the effect of NOS inhibitors on the parenchymal eosinophilic infiltration, mice were killed on day 23. Deparaffinized slides were dipped in alcoholic Congo red solution (0.5% in 50% ethanol) for 30 min, washed with distilled water, and counterstained with Harry’s hematoxylin. This staining led to a specific orange color of eosinophilic granules (26). For each animal, 30 high power fields (magnification x200) were selected randomly away from large blood vessels and airways for parenchymal eosinophil counting. Peribronchial and perivascular eosinophil counts were done in 20 high power fields. Measurement of length of basement membrane and parenchymal area were done using a graticulated ocular. Slide analysis was performed in blind fashion, and the observer coefficient of variation for repeated estimation was less than 3%.
Chemokine expression
For mRNA determination, lungs were taken from four mice 6 h after the challenge and homogenized separately in TRIzol reagent (Life Technologies, Paisley, Scotland). Total RNA were prepared according to the instructions of the supplier. Chemokine RNA expression was analyzed using the multiprobe ribonuclease protection assay system mCK-5 (PharMingen, Oxford, U.K.). In a separate experiment, the levels of chemokine protein were determined in whole lung homogenate (27) by specific ELISA. Briefly, lungs from five to seven mice per group were individually homogenized in 2 ml of PBS containing 0.05% Triton X-100 and a protease inhibitor cocktail (Complete; Boehringer Mannheim, Lewes, U.K.). The resulting supernatant was isolated by centrifugation (10,000 x g for 20 min), and eotaxin, monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) levels were measured using commercially available ELISAs according to the instructions of the supplier (R&D Systems, Abingdon, U.K.).
Determination of NO donor-induced chemokine production from lung fibroblasts
Cells were isolated from naive mice using a procedure modified from Phan et al. (28). Lungs were perfused with PBS/heparin (100 U/ml) via the right ventricle until they were pale. The lung was then minced into small pieces before being incubated for 30 min at 37°C with a mixture of 2.5 mg/ml trypsin and 1 mg/ml collagenase IV in DMEM. The resulting solution was filtered through a 70-µm cell stainer, and cells were washed twice with culture medium (DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin) before being seeded onto 25-cm2 culture flasks. Experiments were undertaken with fibroblasts obtained from the third passage. At this passage, the confirmation of the cells as fibroblasts was determined by morphologic appearance at confluence and by immunolabeling with an antifibroblast Ab (29). Briefly, confluent cells were fixed with 3% formaldehyde solution for 5 min and permeabilized in ice-cold methanol for 2 min. The primary Ab (1/100; Biogenesis, Poole, U.K.) was incubated for 90 min before an anti-rat IgG-FITC Ab for 30 min (1/300; Sigma). Under these conditions, more than 95% of the cells were positively stained.
Confluent lung fibroblasts cultured in 48-well plates were incubated,
in triplicate, with medium or increasing concentration of hydroxylamine
in medium (100–1000 µM; Sigma). After a 24-h incubation at 37°C in
a 5% CO2 atmosphere, chemokine levels in the
supernatant were determined using commercially available ELISAs
according to the instructions of the supplier (R&D Systems).
Recombinant murine IL-4 and TNF- (20 ng/ml; R&D Sytems) were used as
positive control. Cell viability was determined with the MTT viability
kit (Boehringer Mannheim, Rotkreuz, Switzerland) as described
above.
Statistics
Data are expressed as mean ± SEM. Statistical comparisons were performed using an ANOVA and then a Dunnett’s test. A p value of less than 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In sensitized mice challenged with PBS, there was a very weak
staining for iNOS at 3 days postchallenge. Positive cells were mainly
localized beneath the epithelium of most of the airways. Slight
immunoreactivity was also observed in some of the macrophages (Fig. 1
A). In contrast, at the same
time point sensitized mice challenged with OVA showed a strong positive
iNOS staining. Immunoreactivity was present in the cells among the
inflammatory infiltrate located beneath the basement membrane. The
staining of basally located cells observed in the PBS-challenged mice
was greatly enhanced. However, it was not possible to clearly identify
the type of cells expressing iNOS immunoreactivity (Fig. 1B). The negative control sections, in which the
anti-iNOS Ab was replaced by control rabbit IgG, showed a weak
diffuse staining not localized to any tissue structure (data not
shown). To confirm the immunostaining findings, we also measured NOS
activity in lung preparations from mice sacrificed 3 days after the
challenge. As shown in Fig. 2, OVA
challenge induced a 2-fold increase in NOS activity compared with that
of PBS-challenged mice. The majority of this increased activity was
calcium-independent.
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To study the possible involvement of NO in the commitment phase of
Th2 cells, mice were treated twice a day from the second OVA i.p.
injection (boost) until the day before challenge and were sacrificed at
3 days postchallenge. We first used the nonselective NOS inhibitor
L-NAME (100 and 300 mg/kg). As shown in Table I, both L-NAME and
NG-nitro-D-arginine
methyl ester (D-NAME), the inactive enantiomer,
had no significant effect on BAL cell number (eosinophils, macrophages,
neutrophils, and lymphocytes) in comparison with vehicle-treated mice.
In addition, total serum IgE level (Table I) and peripheral blood cell
counts (data not shown) were unaffected by both compounds. Based on
these data, NO did not appear to be involved during this period, which
is why the effect of specific iNOS inhibitors was not further
investigated.
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production decreased (1282 ±
85.5 U/ml) compared with PBS-treated mice (IL-4, 12.3 ± 0.9 U/ml;
IL-5, 710.6 ± 32.5 U/ml; IFN-, 1827 ± 203.6 U/ml;
p < 0.05). D-NAME (100 mg/kg)
had no effect on lung T cell cytokine production (IL-4, 16.3 ±
3.5 U/ml; IL-5, 712.7 ± 125.7 U/ml; IFN-, 2072 ± 457.1
U/ml). Effects of NOS inhibitor treatment from day 20 to day of sacrifice
In a second set of experiments, the mice were treated from the day
before challenge (day 20) until the day of sacrifice. At 3 days
postchallenge, L-NAME (100 and 300 mg/kg) or the specific
iNOS inhibitors (20 mg/kg EIT and 4 mg/kg AMT) had no significant
effect on the number of macrophages and lymphocytes in the BAL (data
not shown). In contrast, all three compounds significantly inhibited
the number of BAL eosinophils and neutrophils (Fig. 3). Compared with AMT and EIT,
L-NAME was statistically less potent at 100 mg/kg,
inhibiting eosinophilia by 37% and neutrophilia by 38%
(p < 0.05). However, at a dose of 300 mg/kg,
L-NAME was as potent as the two specific iNOS
inhibitors, inhibiting eosinophilia by 50% and neutrophilia by 69%.
D-NAME (100 and 300 mg/kg) had no effect on any
of the cell types counted. At 2 days postchallenge using
L-NAME (300 mg/kg) and AMT (4 mg/kg), similar
effects on perivascular, peribronchial, and parenchymal eosinophilic
infiltration were observed, whereas D-NAME (300
mg/kg) had no effect (Table II).
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). In
contrast to the BAL cell count, no change in any of the cell types in
the peripheral blood was observed at 3 days postchallenge in comparing
NOS inhibitor-treated mice with control mice (data not shown). Total
serum IgE level was also unaffected by NOS inhibitors (data not
shown).
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(617 ± 130.2 U/ml) production decreased in mice treated
with AMT (4 mg/kg) compared with the control group (IL-4, 10.5 ±
1.1 U/ml; IL-5, 949.4 ± 85.4 U/ml; IFN-, 1241.7 ± 280.7
U/ml; p < 0.05). Effect of hydroxylamine on survival of purified BAL eosinophils
It has been reported that NO donors can inhibit the death of human blood eosinophils in vitro (18). We studied the possible survival effect of the NO donor hydroxylamine on purified BAL eosinophils. After 48 h of culture, there was no significant difference in the cell counts of nontreated eosinophils and eosinophils treated with 300 µM of hydroxylamine. The OD values (570–660 nm) were 0.251 ± 0.009 and 0.259 ± 0.019, respectively.
Effect of AMT on lung chemokine expression in vivo
To evaluate whether iNOS inhibitors control chemokine production,
mice were treated with AMT from day 20, and the lung mRNA expression
was determined by ribonuclease protection assay 6 h after the
challenge. Compared with PBS-challenged mice, OVA challenge induced a
significant increase in eotaxin, MIP-2, MCP-1, and T cell activation
gene 3 mRNA expression. RANTES, which was constitutively expressed in
PBS-challenged mice, was not up-regulated after OVA challenge (Fig. 5). OVA-induced increase in MIP-2 and
MCP-1 was reduced significantly in the lung of AMT-treated mice,
whereas eotaxin and T cell activation gene 3 expression were not
altered (Fig. 5). At the protein levels, eotaxin, MCP-1, and MIP-2 lung
levels were significantly increased over baseline 6 h after OVA
challenge. By 24 h, compared with the 6-h time point, eotaxin
levels still increased, whereas MCP-1 levels did not change and MIP-2
reached control background levels. OVA-induced increase in MIP-2 and
MCP-1 was significantly inhibited in AMT-treated mice. Eotaxin levels
were reduced, but this was not significant (Fig. 6).
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To better understand how NO could be involved in the up-regulation
of chemokine expression, we studied whether a NO donor, hydroxylamine,
could induced eotaxin, MIP-2, and MCP-1 release from lung fibroblasts.
After 24 h, 7.3 ± 0.2 ng/ml of MCP-1 and 80 ± 3.9
pg/ml of MIP-2 was detected in culture supernatants of untreated
fibroblast. Hydroxylamine induced a dose-dependent release of both
MIP-2 and MCP-1, with a maximal effect at 600 µM for both chemokines.
At this maximum dose, hydroxylamine induced a 3- and 7-fold increase
over basal levels for MCP-1 and MIP-2, respectively (Fig. 7). No detectable levels of eotaxin could
be measured in untreated cells. Furthermore, hydroxylamine did not
induce eotaxin synthesis. This was not a result of the inability of
these cells to produce eotaxin because, after IL-4 treatment,
269.1 ± 13.8 pg/ml of eotaxin was detected in the culture
supernatants (Fig. 7). Cell viability, as determined by MTT assay, was
not affected by any of the treatment (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the airways of asthmatic patients (9) or in rodent lung after allergen challenge (30, 32, 33), iNOS expression and/or enzymatic activity are increased. We have clearly shown that OVA-challenged mice present a 2-fold increase in total lung NOS activity. The majority of this activity was calcium-independent, suggesting the involvement of iNOS. These results were reinforced by immunostaining studies in which lung sections from PBS-challenged mice showed no positive staining. In contrast, a positive immunostaining, which is associated with the alveolar macrophages and granulocytes, was evident in animals challenged with OVA. This pattern of staining contrasts with the one observed in asthmatic lungs, showing mainly epithelial staining (9). This discrepancy could be related to species variation. Alternatively, the stimuli used in the present study (one allergen provocation) may not be powerful enough to induce iNOS expression in the epithelium. Indeed, when using repeated challenge (daily challenge for 5 days), a positive staining for iNOS in the airway epithelium was observed (unpublished data). Altogether, we can assume that the up-regulation of iNOS is associated with an increased release of NO in the airways. These observations together with the fact that the specific and potent iNOS inhibitors, AMT and EIT (20, 21), inhibited granulocyte lung infiltration suggested that the production of NO after allergen challenge in our model was mainly due to the activation of iNOS.
NO is now emerging as a potential modulator of T cell responses
(34). In cloned murine T cell lines, it has been shown
that NO was an inhibitory factor of Th1 cell development, whereas it
had no effect on Th2 cells (35). From these results, it
was postulated that NO could play an important role in amplifying and
perpetuating the Th2 cell-mediated inflammatory response in asthma
(17). This hypothesis predicts that inhibition of NOS
would result in a decrease of the eosinophilic inflammation. However,
our results demonstrating the inhibition of both eosinophilic and
neutrophilic airway infiltration may not directly support this
hypothesis. First, inhibition of inflammatory cell infiltration was
evident only when mice were treated with NOS inhibitors from the
challenge to the sacrifice but not from the second immunization to the
challenge, which is known to be the commitment period for T cells in
this model (19). Second, purified lung T cells showed the
same profile of cytokine production with both treatments, namely an
increase in Th2 type of cytokines and a decrease in Th1 type. The
modulation of T cell cytokines toward Th1 by NO contrasts with previous
findings (35). However, the effect of NO in vivo on the
immune system response appears to vary in different systems. In a
murine malaria model, IFN- production from spleen cells was
significantly enhanced when the mice were treated with a NOS inhibitor
(35). In contrast, in a carrageenin-induced edema murine
model, it was shown that lymph node T cells from animals treated with
NOS inhibitor produce less IFN- and more Th2 type of cytokines
(e.g., IL-10) (36). The mechanism by which NO can affect T
cell activation and cytokine production is at present unknown.
However, the discrepancy observed in vivo could be explained by a
dose-dependent effect of NO in these various models
(36).
Both IL-4 and IL-5 are key cytokines in the development of the asthmatic airway inflammation. IL-4 is of critical importance for IgE production, and IL-5 is involved in eosinophil maturation and survival (37). One can assume that increase of IL-4 and IL-5 should result in an amplification of the granulocyte infiltration of the airways. However, despite the increase in IL-4 and IL-5 production, we observed an inhibition of the airway’s inflammatory cell infiltration in animals treated with NOS inhibitors from the day of challenge. This result seems to contradict the established role of these two cytokines. However, NO induces a variety of responses that may affect other aspects of the inflammatory process. For instance, NO has been described as a survival factor for human blood eosinophils (18). However, we were not able to prolong the survival of murine BAL eosinophils with NO donors. This discrepancy could be explained by the fact that BAL eosinophils are highly activated, compared with blood eosinophils, by transendothelial and extravascular migration into the mucosal tissue (38). Moreover, this different activation state could explain the different sensitivity to NO. Nevertheless, we were able to exclude a possible role of NO in the survival of BAL eosinophils in our model.
The fact that NOS inhibitors inhibited granulocyte infiltration only when applied from Ag challenge to sacrifice suggests that NO is involved not in the first step of the inflammatory reaction but rather during the final stage of this process (such as during endothelial transmigration or chemotaxis). The endothelial expression of adhesion molecules such as ICAM-1 and VCAM-1 is critical for the accumulation in vivo of eosinophils in the lungs (39, 40). Although it has been shown that NOS inhibition may modulate the expression of these molecules in vitro (41, 42), these observations do not seem to be confirmed in vivo. As such, in an experimental model of colitis, iNOS deficiency and/or NOS inhibition do not alter the increased expression of both ICAM-1 (43) and VCAM-1 (44). However, in vitro and in vivo data have shown that NO contributes to both neutrophil (45, 46) and eosinophil (47) chemotaxis. Therefore, we speculated that the inhibition of the leukocyte infiltration observed in our model was not because of a modulation of the adhesion process to the endothelium, but rather that it was because of a decrease in the chemotaxis activity in the lung mucosa. To the end we studied the effect of AMT treatment on lung chemokine expression after allergen challenge. Although eosinophilic chemokines like RANTES or eotaxin were unaffected by AMT treatment, others like MIP-2 (a chemoattractant for neutrophils) and MCP-1 (which is primarily involved in the recruitment of mononuclear cells) were clearly reduced after AMT treatment. This decrease in MCP-1 mRNA and protein expression is interesting because this chemokine has been shown to be a critical player in a similar murine model of OVA-induced lung inflammation (48). In this elegant study using neutralizing Abs, it was clearly shown that, although eotaxin is important for eosinophil recruitment after the Ag provocation, MCP-1 (even in the presence of eotaxin) is critical throughout the development of the inflammatory response (48).
Although there is increasing evidence that NO could be involved in the up-regulation of cytokines (49, 50), little is known about the effect of NO on chemokine production. Based on our in vivo results, we could not differentiate whether NO induced a direct up-regulation of chemokine production or if it increased the survival of chemokine-producing cells. To address this question, we studied whether a NO donor, hydroxylamine, could induce chemokine release from primary cultures of murine lung fibroblasts, a cell type known to produce MCP-1 (51), MIP-2 (52), and eotaxin (53). In line with the in vivo data, we have shown that hydroxylamine dose dependently up-regulated MCP-1 and MIP-2 release and that it had no effect on eotaxin. This strongly suggests that, in our model, NO is directly involved in the specific up-regulation of MCP-1 and MIP-2 rather than acting as a survival factor for chemokine-producing cells.
In conclusion, we have shown that, in this murine model of allergic asthma, iNOS promotes airway lung inflammation via direct up-regulation of chemokine expression. Our data suggest that NO could be involved in the chemotactic process toward granulocytes in the lung mucosa. Finally, the present results support the idea that the development of specific iNOS inhibitors may represent a novel therapeutic approach for asthma (16, 17).
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Acknowledgments |
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Footnotes |
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2 Abbreviations used in this paper: NOS, NO synthase; iNOS, inducible NO synthase; EIT, S-ethylisothiourea; AMT, 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine; L-NAME, NG-nitro-L-arginine methyl ester; BAL, bronchoalveolar lavage; MCP-1, monocyte chemoattractant protein-1; MIP-2, macrophage inflammatory protein-2; D-NAME, NG-nitro-D-arginine methyl ester.
Received for publication October 15, 1999. Accepted for publication April 26, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 C. M. Prado, E. A. Leick-Maldonado, V. Arata, D. I. Kasahara, M. A. Martins, and I. F. L. C. Tiberio Neurokinins and inflammatory cell iNOS expression in guinea pigs with chronic allergic airway inflammation Am J Physiol Lung Cell Mol Physiol, April 1, 2005; 288(4): L741 - L748. [Abstract] [Full Text] [PDF]
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P. Keshavan, T. L. Deem, S. J. Schwemberger, G. F. Babcock, J. M. Cook-Mills, and S. D. Zucker Unconjugated Bilirubin Inhibits VCAM-1-Mediated Transendothelial Leukocyte Migration J. Immunol., March 15, 2005; 174(6): 3709 - 3718. [Abstract] [Full Text] [PDF]
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 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]
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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]
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G. C. Koo, K. Shah, G. J. F. Ding, J. Xiao, R. Wnek, G. Doherty, X. C. Tong, R. B. Pepinsky, K.-C. Lin, W. K. Hagmann, et al. A Small Molecule Very Late Antigen-4 Antagonist Can Inhibit Ovalbumin-induced Lung Inflammation Am. J. Respir. Crit. Care Med., May 15, 2003; 167(10): 1400 - 1409. [Abstract] [Full Text] [PDF]
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I. Jibiki, S. Hashimoto, S. Maruoka, Y. Gon, A. Matsuzawa, H. Nishitoh, H. Ichijo, and T. Horie Apoptosis Signal-Regulating Kinase 1-Mediated Signaling Pathway Regulates Nitric Oxide-Induced Activator Protein-1 Activation in Human Bronchial Epithelial Cells Am. J. Respir. Crit. Care Med., March 15, 2003; 167(6): 856 - 861. [Abstract] [Full Text] [PDF]
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 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]
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S. Ferretti, O. Bonneau, G. R. Dubois, C. E. Jones, and A. Trifilieff IL-17, Produced by Lymphocytes and Neutrophils, Is Necessary for Lipopolysaccharide-Induced Airway Neutrophilia: IL-15 as a Possible Trigger J. Immunol., February 15, 2003; 170(4): 2106 - 2112. [Abstract] [Full Text] [PDF]
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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]
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 K. Minamoto and D. J. Pinsky Recipient iNOS but Not eNOS Deficiency Reduces Luminal Narrowing in Tracheal Allografts J. Exp. Med., November 18, 2002; 196(10): 1321 - 1333. [Abstract] [Full Text] [PDF]
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L. R. Watkins and S. F. Maier Beyond Neurons: Evidence That Immune and Glial Cells Contribute to Pathological Pain States Physiol Rev, October 1, 2002; 82(4): 981 - 1011. [Abstract] [Full Text] [PDF]
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R. A. Skidgel, X.-p. Gao, V. Brovkovych, A. Rahman, D. Jho, S. Predescu, T. J. Standiford, and A. B. Malik Nitric Oxide Stimulates Macrophage Inflammatory Protein-2 Expression in Sepsis J. Immunol., August 15, 2002; 169(4): 2093 - 2101. [Abstract] [Full Text] [PDF]
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S. Yang, V. A. Porter, D. N. Cornfield, C. Milla, A. Panoskaltsis-Mortari, B. R. Blazar, and I. Y. Haddad Effects of oxidant stress on inflammation and survival of iNOS knockout mice after marrow transplantation Am J Physiol Lung Cell Mol Physiol, October 1, 2001; 281(4): L922 - L930. [Abstract] [Full Text] [PDF]
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