help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Feder, L. S.
Right arrow Articles by Kung, T. T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Feder, L. S.
Right arrow Articles by Kung, T. T.
Am. J. Respir. Cell Mol. Biol., Volume 17, Number 4, October 1997 436-442

Role of Nitric Oxide on Eosinophilic Lung Inflammation in Allergic Mice

L. S. Feder, D. Stelts, R. W. Chapman, D. Manfra, Y. Crawley, H. Jones, M. Minnicozzi, X. Fernandez, T. Paster, R. W. Egan, W. Kreutner, and T. T. Kung

Schering-Plough Research Institute, Kenilworth, New Jersey


    Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Nitric oxide (NO) is an important mediator of inflammatory reactions and may contribute to the lung inflammation in allergic pulmonary diseases. To assess the role of NO in pulmonary inflammation, we studied the effect of four nitric oxide synthase (NOS) inhibitors, N-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, NG-monomethyl-L-arginine (NMMA) and L-N6-(1-Iminoethyl) lysine (L-NIL), on the influx of eosinophils into the bronchoalveolar lavage (BAL) fluid and lung tissue of antigen-challenged allergic mice. We also analyzed lung tissues for the presence of steady state mRNA for inducible nitric oxide synthase (iNOS) and iNOS protein. Furthermore, BAL fluid and serum were analyzed for their nitrite content. B6D2F1/J mice were sensitized to ovalbumin (OVA) and challenged with aerosolized OVA. The NOS inhibitors were given 0.5 h before and 4 h after the antigen challenge. OVA challenge induced a marked eosinophilia in the BAL fluid and lung tissue 24 h after challenge. The OVA-induced pulmonary eosinophilia was significantly reduced by L-NAME (10 and 50 mg/kg, intraperitoneally [i.p.]). The inactive isomer, D-NAME (50 mg/kg, i.p.) had no effect. When mice were treated with L-NAME (20 mg/kg, i.p.) and an excess of NOS substrate, L-arginine (200 mg/kg, i.p.), the OVA-induced pulmonary eosinophilia was restored. Treatment with aminoguanidine (0.4  -50 mg/kg, i.p.) also reduced the pulmonary eosinophilia. Treatment with NMMA (2-50 mg/kg, i.p.) partially reduced the eosinophilia, but L-NIL (10-50 mg/kg, i.p.), a selective iNOS inhibitor, had no effect. L-NAME had no effect on the reduction of eosinophils in the bone marrow following OVA challenge to sensitized mice. OVA challenge to sensitized mice had no effect on iNOS protein expression or iNOS mRNA in the lungs or on the levels of nitrite in the BAL fluid. These results suggest that NO is involved in the development of pulmonary eosinophilia in allergic mice. The NO contributing to the eosinophilia is not generated through the activity of iNOS nor does NO contribute to the efflux of eosinophils from the bone marrow in response to antigen challenge. It is speculated that after antigen challenge, the localized production of NO, possibly from pulmonary vascular endothelial cells, is involved in the extravasation of eosinophils from the circulation into the lung tissue.


    Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Nitric oxide (NO) is an important mediator of biological functions in the lung and regulates airway smooth muscle contractility (1), ventilation/perfusion relationships (2), and secretion of mucus from airway mucus glands (4). NO is also an important mediator of inflammatory responses in the lungs and produces this effect by the formation of reactive nitrogen products that are released from a variety of inflammatory cells (5). Nitric oxide synthase (NOS) is a key enzyme in the formation of NO and both the constitutive (cNOS) and inducible (iNOS) isoforms have been described in human alveolar and bronchial epithelial cells (8). The activity of cNOS is calcium dependent and can be increased 2-3-fold by agents such as bradykinin and calcium ionophore (9, 10). The generation of NO by cNOS is rapid, occurring within seconds (9, 10). On the other hand, the activity of iNOS is calcium-independent and can be increased up to 20-fold by activation with cytokines or endotoxin (11, 12). Although maximal induction of iNOS requires several hours, cells will produce NO over a period of several days (11, 12). Increased expression of iNOS has been found in the airway epithelium of human asthmatics (13) and NO is increased in the exhaled air of these subjects when compared to normals (14, 15). In addition, iNOS activity is increased in the lung tissue of sensitized and challenged guinea pigs (16, 17), suggesting that NO is important in the pathogenesis of allergic lung disorders.

Pulmonary eosinophilic inflammation is a characteristic feature of inflammatory airway diseases, such as asthma (18, 19). The eosinophil granule contains several proteins, which have direct toxic effects on the airway cells and may result in the bronchial hyperresponsiveness of asthmatic airways (18). Recently, we reported that pulmonary eosinophilia and lung tissue damage was induced in mice that were sensitized and challenged with aerosolized ovalbumin (OVA) (21). Furthermore, the pulmonary eosinophilia and lung tissue damage is ameliorated by treatment with anti-inflammatory agents such as corticosteroids and antibodies to interleukin-5 (IL-5) (21). Although nitric oxide is recognized as a contributor to pulmonary inflammation after allergen challenge (16, 17), its role in the recruitment of eosinophils into the lungs is unknown.

In this study, we investigated the effect of four NOS inhibitors, N-nitro-L-arginine methyl ester (L-NAME), aminoguanidine, NG-monomethyl-L-arginine (NMMA), and L-N6-(1-Iminoethyl) lysine (L-NIL), on the influx of eosinophils into the bronchoalveolar lavage (BAL) fluid and lung tissue of allergen-challenged mice. We also analyzed the lungs for expression of the iNOS protein and steady state mRNA. Nitrite levels in the serum and BAL fluid were also measured following antigen challenge.

    Materials and Methods
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mice and Sensitization

Male B6D2F1/J mice (Jackson Labs, Bar Harbor, ME) weighing 20-25 g were sensitized by an intraperitoneal (i.p.) injection of 0.5 ml alum-precipitated antigen containing 15 µg of OVA adsorbed to 2 mg of aluminum hydroxide (alum) gel in 0.9% saline vehicle. A booster injection of this alum-ovalbumin mixture was given 5 days later. Nonsensitized control animals received the alum gel only.

Antigen Challenge and Collection of Fluids and Specimens

Twelve days after sensitization, the mice were placed in a Plexiglas chamber and challenged with aerosolized OVA (0.5%) for 1 h duration both in the morning and afternoon of a single day. The aerosolized OVA was produced by an ultrasonic nebulizer (model Ultra-Neb 99; DeVilbiss, Somerset, PA). At either 6 or 24 h after antigen challenge, the mice were killed by CO2 asphyxiation and necropsy was performed to collect samples of BAL fluid, lung tissue, blood and bone marrow aspirate. The total cell numbers in the BAL fluid and bone marrow aspirate were measured with a standard hemocytometer and differential counts were made from cytospin preparations (21). Histological evaluation of eosinophil accumulation in the lungs was assessed using previously described methodologies (21). The number of eosinophils in five randomly selected areas of the peribronchial regions of the lung were counted at ×400 magnification and expressed as the number of eosinophils per high power field.

The NOS inhibitors (L-NAME, aminoguanidine, NMMA, and L-NIL) were injected i.p. 30 min before and again 4 h after the inhaled OVA challenge. In studies involving combined L-arginine and L-NAME, the L-arginine was injected i.p. immediately before the injection of the L-NAME.

RNA Isolation, cDNA Synthesis, and Competitive Polymerase Chain Reactions

Freshly isolated lung tissue or BAL cells from 3-4 mice were pooled, and total RNA was isolated using Tri-Reagent (Molecular Reagents, Inc., Cincinnati, OH). A second extraction with the Tri-Reagent was performed prior to isopropanol precipitation of the RNA. The isolated RNA was treated with DNase I to degrade any residual chromosomal DNA. Total RNA preparations were subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) analysis.

cDNA was synthesized from 2 µg total RNA primed with oligo (dT)12-18 using the Superscript II Preamplification System (GibcoBRL, Grand Island, NY). The cDNA was amplified in a competitive PCR using target-specific primers. The competitive PCR analysis was based on the PCR MIMIC System (Clontech Laboratories, Inc., Palo Alto, CA). The MIMICs were purchased from Clontech or synthesized using the MIMIC construction kit (Clontech Laboratories).

For semiquantitative competitive PCR analysis, a constant amount of the target-specific PCR MIMIC was added to each PCR reaction. The cDNA and MIMIC were co-amplified in a 50-µl volume using 2-5 µl of cDNA (equivalent to 200-500 ng of RNA) and the calculated amount of MIMIC. The samples were subjected to a hot start at 94°C for 1 min, amplified for 35 cycles at 94°C for 1 min, 60°C for 1 min, 72°C for 1.5 min, followed by a final incubation at 72°C for 2 min in a Gene Amp PCR System 9600 (Perkin Elmer Cetus, Foster City, CA).

The oligonucleotides corresponding to the sense and antisense strands, respectively, for each target gene are as follows: iNOS: 5'-CTGGCAGCAGCGGCTCCA-TGACT-CC-3' and 5'-AGCCTCGTGGCTTTGGGCTCCTCCA-3', and G3PDH: TGAAGGTCGGTGTGAACGGATTTG-GC-3' and 5'-CATGTAGGCCATGAGGCCACCAC-3'. The primers spanned introns and were either purchased from Clontech or synthesized on an Applied Biosystems DNA synthesizer and purified using oligonucleotide purification columns (Applied Biosystems, Foster City, CA).

Semiquantitative analysis of the PCR products was also performed. The target-gene and MIMIC PCR products were separated by electrophoresis on a 2% agarose gel containing ethidium bromide. The gels were photographed, and the yield of PCR product for the target gene and the PCR MIMIC was quantified via whole-band analysis using the GS-670 Imaging Densitometer (Bio Rad Laboratories, Hercules, CA). The relative levels of the target gene in each sample are normalized to the G3PDH.

Western Blot Analysis of iNOS

Lung tissue isolated from treatment groups was homogenized in 1 ml of 50 mM Tris-HCl buffer, pH 7.8, containing 150 mM NaCl, 1 mM EDTA (ethylenediaminetetraacetic acid), 1 mM sodium vanadate, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride (PMSF). The samples were transferred to eppendorf tubes and centrifuged at high speed in a microcentrifuge for 10 min at 4°C to remove insoluble material. Protein content was determined using the method of Bradford (24) with bovine serum albumin as the standard. Equal amounts of protein were added to a 0.1 M Tris buffer containing 50 µM dithiothreitol, 0.01% bromophenol blue, 1% sodium dodecyl sulfate (SDS) and 10% glycerol and boiled for 5 min. Proteins (30 µg/lane) were separated on a 7.5% SDS polyacrylamide gel, transferred to nitrocellulose paper and probed with a rabbit antibody against purified inducible nitric oxide synthase from cytokine-induced mouse macrophages (Alexis Corporation, San Diego, CA). Antibody binding was detected using an amplified alkaline phosphatase immuno blot kit (Bio Rad Laboratories, Hercules, CA) according to the manufacturer's recommendation. Samples of mouse macrophage lysates stimulated with interferon-gamma (IFN-gamma ) and lipopolysaccharide (LPS) were used as positive controls (Transduction Laboratories, Lexington, KY).

Nitrite Assay

Nitrite in the serum and BAL fluid was determined by a spectrophotometric method based on the Griess reaction (25). Prior to analysis, nitrate in the samples was reduced to nitrite using the enzyme nitrate reductase (Boehringer Mannheim Corp., Indianapolis, IN) 0.1 units/ml, in the presence of 50 µM NADPH, 5 µM flavin adenine dinucleotide (FAD), 6 µg/ml lactate dehydrogenase, and 0.2 mM sodium pyruvate. A total of 100 µl of sample was mixed with 200 µl of Griess' reagent (1% sulfanilamide, 0.1% naphthylenediamide-dihydrochloride, and 2.5% H3PO4) and incubated at room temperature for 5 min. Nitrite concentrations in the samples were determined using different concentrations of sodium nitrite as a standard curve. The reaction was read on a plate reader at a wavelength of 570 nm.

Reagents

Ovalbumin and aluminum hydroxide gel were obtained from Reheis Inc. (Berkeley Heights, NJ). Ovalbumin was dissolved in 0.9% saline for aerosolization. L-NAME, D-NAME, NMMA, aminoguanidine, and L-arginine were obtained from Sigma Chemical Co. (St. Louis, MO). L-NIL was purchased from Cayman Chemical Co. (Ann Arbor, MI).

Statistical Analysis

Statistically significant differences between groups were determined using an analysis of variance and Fisher's protected least significant difference program (Statview version 4.0; ABACUS Concepts, Berkeley, CA). A P < 0.05 was considered statistically significant.

Animal Care and Use

This study was done in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act in a program approved by the American Association for the Accreditation of Laboratory Animal Care.

    Results
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of NOS Inhibitors on Pulmonary Eosinophilia

There were increased numbers of eosinophils in the BAL fluid and peribronchial regions of the lungs of sensitized mice 24 h after OVA challenge (Figure 1). Sensitized mice, treated with L-NAME (2-50 mg/kg, i.p.), given 30 min before and 4 h after OVA challenge, had reduced numbers of eosinophils in the BAL fluid and lung tissue compared with sensitized, challenged mice treated with i.p. saline or the inactive isomer, D-NAME (50 mg/kg, i.p.). Statistically significant inhibition occurred at 10 mg/ kg (Figure 1). Comparable results were obtained with another NOS inhibitor, aminoguanidine, that significantly reduced the number of eosinophils appearing in the BAL fluid of sensitized, challenged mice at 10 and 50 mg/kg (Figure 2). In addition, NMMA (2-50 mg/kg, i.p.), caused a partial (approximately 40% at 10 and 50 mg/kg), but not a statistically significant inhibition in the BAL eosinophilia of sensitized, challenged mice (data not shown). In contrast, the selective iNOS inhibitor, L-NIL (10-50 mg/ kg, i.p.), had no effect on the recruitment of eosinophils into the lungs of sensitized, challenged mice (Figure 3).


View larger version (31K):
[in this window]
[in a new window]
  		Figure 1.   Effect of L-NAME on the eosinophil number in the BAL and lung tissue of allergic mice. BAL fluid and tissue were collected from allergic mice 24 h after challenge. L-NAME and its enantiomer, D-NAME, were administered i.p. 0.5 h before and 4 h after the first antigen challenge. Values represent the mean ± SEM (n of 10-12 per group). *Statistically significant from the sensitized control group (P =< 0.05).


View larger version (22K):
[in this window]
[in a new window]
  		Figure 2.   Effect of aminoguanidine on the eosinophil number in the BAL of allergic mice. BAL fluid was collected from allergic mice 24 h after challenge. Aminoguanidine was administered i.p. 0.5 h before and 4 h after the first antigen challenge. Values represent the mean ± SEM (n of 10-12 per group). *Statistically significant from the sensitized control group (P =< 0.05).


View larger version (20K):
[in this window]
[in a new window]
  		Figure 3.   Effect of L-NIL on the eosinophil number in the BAL of allergic mice. BAL fluid was collected from allergic mice 24 h after challenge. L-NIL was administered i.p. 0.5 h before and 4 h after the first antigen challenge. Values represent the mean ± SEM (n of 10-12 per group). *Statistically significant from the sensitized control group (P =< 0.05).

Treatment of sensitized mice with L-arginine (200 mg/ kg, i.p.), a substrate for nitric oxide synthase, had no effect on the number of BAL eosinophils of challenged mice, while L-NAME (20 mg/kg, i.p.) produced the expected reduction in BAL eosinophils (Figure 4). However, when L-arginine (200 mg/kg, i.p.) was combined with L-NAME (20 mg/kg, i.p.), the inhibitory effect of L-NAME on the BAL eosinophilia was abolished (Figure 4).


View larger version (21K):
[in this window]
[in a new window]
  		Figure 4.   Effect of L-arginine and L-NAME on the eosinophils in the BAL of allergic mice. BAL fluid was collected from allergic mice 24 h after challenge. L-arginine and L-NAME were administered i.p. 0.5 h before and 4 h after the first antigen challenge. Values represent the mean ± SEM (n of 10-12 per group). *Statistically significant from the sensitized control group (P =< 0.05).

OVA challenge to sensitized mice decreased the number of eosinophilic cells in the bone marrow 24 h after challenge (Figure 5). The decrease in bone marrow eosinophils in OVA-challenged mice was unchanged by treatment with L-NAME (10 mg/kg, i.p.).


View larger version (34K):
[in this window]
[in a new window]
  		Figure 5.   Effect of L-NAME on the bone marrow eosinophils of allergic mice. Bone marrow aspirates were collected from allergic mice 24 h after challenge. L-NAME was administered i.p. 0.5 h before and 4 h after the first antigen challenge. Values represent the mean ± SEM (n of 10-12 per group). *Statistically significant from sensitized control group (P =< 0.05).

Measurement of iNOS Protein, mRNA, and Nitrite

To determine whether allergen challenge increased the production of iNOS enzyme in the lungs and BAL cells, we measured the protein levels of iNOS enzyme and the steady state mRNA for iNOS in the lungs of sensitized OVA-challenged mice. Sensitization and challenge had no effect on iNOS protein expression in the lung tissue measured 24 h after OVA challenge (Figure 6). Steady state levels of iNOS mRNA were also not different between normal, nonsensitized or sensitized and challenged groups measured 6 and 24 h after OVA challenge (Figure 7). Furthermore, there was no iNOS mRNA expression in the cellular fraction of the BAL fluid of sensitized, challenged mice (data not shown). Interestingly, even lung tissue isolated from normal mice expressed levels of iNOS protein and mRNA (Figures 6 and 7).


View larger version (13K):
[in this window]
[in a new window]
  		Figure 6.   Immunoblot analysis of the expression of iNOS in lung tissue isolated from allergic mice. Lane 1: iNOS positive control; lane 2: normal mice; lane 3: sensitized, challenged mice; lane 4: nonsensitized, challenged mice. Lung tissue was isolated from normal, sensitized, challenged and nonsensitized, challenged mice 24 h after challenge. Lungs from 4 different mice from each group were pooled and homogenized in a Tris buffer containing PMSF. Proteins were resolved on a 7.5% SDS polyacrylamide gel and immunoblotted with an antibody to iNOS.


View larger version (37K):
[in this window]
[in a new window]
  		Figure 7.   RT-PCR analysis of the expression of iNOS mRNA in lung tissue isolated from allergic mice. Lane 1: normal mice, 6 h; lane 2: nonsensitized, challenged mice, 6 h; lane 3: sensitized, challenged mice, 6 h; lane 4: normal mice, 24 h; lane 5: nonsensitized, challenged mice, 24 h; lane 6: sensitized, challenged mice, 24 h; lane 7: 1 Kb ladder. Total RNA was isolated using the Tri reagent from the lungs of normal, nonsensitized challenged and sensitized challenged mice. Lung tissue from 3 mice in each group was collected and pooled 6 or 24 h after challenge. Total RNA was subjected to RT-PCR analysis to determine whether iNOS mRNA was induced by the treatments.

Additional studies were performed measuring the levels of nitrite in the serum and BAL fluid of mice. There was a small but statistically significant increase in serum levels of nitrite in response to antigen sensitization and challenge (Figure 8). In contrast, there was no difference in BAL nitrite levels in the treatment groups at either time point (Figure 8).


View larger version (13K):
[in this window]
[in a new window]
  		Figure 8.   Nitrite levels in serum and BAL fluid isolated from allergic, challenged mice. Blood and BAL fluid were collected from nonsensitized, challenged, and sensitized, challenged mice 6 or 24 h after challenge. Values represent the mean ± SEM (n of 4-8 per group). *Statistically significant from nonsensitized, challenged mice (P <=  0.05).

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Allergen challenge to sensitized mice produces many of the characteristic features of human asthma such as eosinophil infiltration into the airways (21, 26), bronchial hyperresponsiveness (27, 28), and damage of the pulmonary tissues (21, 23). In this study, we investigated the possible role of endogenously released NO on the development of pulmonary eosinophilia. Although nonselective nitric oxide synthase inhibitors reduced the number of eosinophils in the BAL fluid and lung tissue of antigen-challenged mice, L-NIL, a selective inhibitor of iNOS (29), did not. In addition, L-NAME failed to reverse the decrease of eosinophils in the bone marrow following sensitization and challenge, suggesting that NO is not important in trafficking eosinophils from the bone marrow into the blood. We also measured the expression of iNOS protein, the steady state mRNA for iNOS in airway tissue, and the levels of nitrite in the serum and BAL fluid. There was no evidence for an increase in pulmonary iNOS in these tissues and fluids of sensitized, antigen-challenged mice. Similar findings have been reported in OVA sensitized and challenged guinea pigs where no detectable increase in NOS activity or mRNA was found in the lungs after antigen challenge even though increased NO was detected in the exhaled air (30).

Nitric oxide synthase is associated with many important physiological systems. In the lungs, NO controls airway smooth muscle contractility (1), ventilation/perfusion relationships (2), and mucociliary function (4, 31). Furthermore, NO is an important mediator of inflammatory responses and this effect is produced by the formation of highly reactive nitrogen species (2, 5). NOS activity can be inhibited by giving compounds like L-NAME, aminoguanidine, L-NMMA, and L-NIL that are analogs of L-arginine (29, 32). We used doses of L-NAME, aminoguanidine, L-NMMA, and L-NIL that have previously been used to block NO formation in other physiological systems (35- 37). L-NAME, aminoguanidine, and L-NMMA inhibited pulmonary eosinophilia, but L-NIL was inactive. We confirmed the specificity of L-NAME for inhibition of NOS by dosing with an excess of the substrate L-arginine and reversing the inhibitory effects of L-NAME on eosinophil recruitment.

Three forms of NOS have been described in mammalian tissues. The two constitutive NOS are found predominantly in endothelial (type III) and nerve cells (type I) (38). Type III NOS is also found in bronchial epithelial, neutrophils, platelets and mast cells (38, 40). The other form of NOS (type II) is inducible, and is found in macrophages, fibroblasts, smooth muscle cells, endothelial cells, epithelial cells, and neutrophils (6, 8, 38, 39, 41). In this study, we found no evidence for increased iNOS in the lungs of sensitized, antigen-challenged mice. We measured the expression of the iNOS protein and the steady state mRNA for iNOS in the lungs and cellular fraction of the BAL fluid to quantify the response. We have previously used these techniques to demonstrate an increase in endogenous NO formation in the lungs of mice in response to provocation with endotoxin (42).

Although we found no increase in the inducible form of NOS in the lung of sensitized and challenged mice, this does not rule out the possibility of a localized NO production. For example, mast cells have the capacity to synthesize NO (38, 39), and mast cells contribute to the development of pulmonary eosinophilia in allergic mice (43, 44). Nitric oxide derived from endothelial cells of lung capillaries and/or bronchial epithelial cells is under control of constitutive endothelial NOS (8, 40, 45) and may be a source of NO. If pulmonary endothelial cells were responsible for the NO production, this could explain why nitrites increased in the serum of sensitized, challenged mice. In this murine model, eosinophils usually aggregate around blood vessels and bronchial airways after antigen challenge (21), which also suggests that NO generated from vascular endothelium and bronchial epithelium may be important sources of NO.

There are several mechanisms by which NO may recruit eosinophils into the lungs following an allergic reaction. Eosinophils are released from the bone marrow into the blood after antigen challenge (21). However, our results suggest that the bone marrow is not an important site of action for NO, because treatment with NOS inhibitors had no effect on the reduction of bone marrow eosinophils in sensitized, challenged mice. These results are in contrast to the site of action of corticosteroids and antibodies to IL-5 that inhibit eosinophil efflux from the bone marrow after antigen challenge (21). Nitric oxide also produces airway microvascular leakage (46) which may augment the migration of eosinophils from the blood into the lungs. Additionally, NO is chemotactic for a variety of cell types including eosinophils (47) and may, thereby, play a role in the recruitment of these cells into the lungs of allergic mice.

Nitric oxide also has an effect on T-cell function and inhibits the proliferation of cloned Th1 cells and their production of IL-2 and IFN-gamma (50). By contrast, Th2 cells neither produce nor are affected by NO (50). IFN-gamma is known to inhibit Th2 cell proliferation (51). Thus, production of large amounts of NO could reduce the secretion of IFN-gamma resulting in an increase of Th2 cell proliferation (52). The important eosinophil-active cytokines that are released from Th2 cells are IL-4 and IL-5 (53). We have previously found increased levels of steady state mRNA for these cytokines in the lungs of sensitized, antigen-challenged mice (26) and treatment with IFN-gamma or antibodies to IL-5 and IL-4 inhibited the antigen-induced pulmonary eosinophilia (22). This raises the possibility that endogenously released NO increases eosinophil recruitment into the lungs by modulating cytokine activity from Th2 cells.

In conclusion, our results demonstrate inhibition by nonselective NOS inhibitors of the pulmonary eosinophilia induced by antigen challenge in allergic mice. This response is not due to an effect on bone marrow precursors because NOS inhibitors do not block eosinophil release from the bone marrow. The NO contributing to the eosinophilia is not generated through the activity of iNOS because the selective iNOS inhibitor, L-NIL, had no effect on eosinophil influx into the lungs. In addition, there was no increase in the level of iNOS protein or mRNA in the lungs or on the levels of nitrite in the BAL fluid. However, serum nitrite levels were increased after OVA challenge. It is speculated that the localized production of NO, possibly from pulmonary vascular endothelial cells, is involved in the extravasation of eosinophils from the circulation into the lung tissue.

    Footnotes

Address correspondence to: Richard W. Chapman, Ph.D., Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033-0539.

(Received in original form November 20, 1996 and in revised form February 18, 1997).

Acknowledgments: The authors thank Ms. Carol Battle for assisting in the preparation of the manuscript.

Abbreviations BAL, bronchoalveolar lavage; L-NAME, N-nitro-L-arginine methyl ester; NMMA, NG-monomethyl-L-arginine; L-NIL, L-N6-(1-Iminoethyl)lysine; NO, nitric oxide; NOS, nitric oxide synthase; OVA, ovalbumin; PCR, polymerase chain reaction.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1. Nijkamp, F. P., and G. Folkerts. 1994. Nitric oxide and bronchial reactivity. Clin. Exp. Allergy 244: 905-914 .

2. Gaston, B., J. M. Drazen, J. Loscalzo, and J. S. Stamler. 1994. The biology of nitrogen oxides in the airways. Am. J. Respir. Crit. Care Med. 149: 538-551 [Abstract].

3. Yan, Z.-Q., K. Kramer, A. Bast, and H. Timmerman. 1994. The involvement of nitric oxide synthase in the effect of histamine on guinea pig airway smooth muscle. Agents Actions 41: 111-112 [Medline].

4. Adler, K. B., B. M. Fischer, H. Li, N. H. Choe, and D. T. Wright. 1995. Hypersecretion of mucin in response to inflammatory mediators by guinea pig tracheal epithelial cells in vitro is blocked by inhibition of nitric oxide synthase. Am. J. Respir. Cell Mol. Biol. 13: 526-530 [Abstract].

5. Jorens, P. G., F. J. VanOverveld, H. Butt, P. A. Vermeire, and A. G. Herman. 1991. L-arginine-dependent production of nitrogen oxides by rat pulmonary macrophages. Eur. J. Pharmacol. 200: 205-209 [Medline].

6. Rimele, T. J., R. J. Sturm, L. M. Adams, D. E. Henry, R. J. Heaslip, B. M. Weichman, and D. Grimes. 1988. Interaction of neutrophils with smooth muscle: identification of a neutrophil-derived relaxing factor. J. Pharmacol. Exp. Ther. 245: 102-111 [Abstract/Free Full Text].

7. Bissonnette, E. Y., C. M. Hogaboam, J. L. Wallace, and A. D. Befus. 1991. Potentiation of tumor necrosis factor-alpha -mediated cytoxicity of mast cells by their production of nitric oxide. J. Immunol. 147: 3060-3065 [Abstract].

8. Asano, K., C. B. E. Chee, B. Gaston, C. M. Lilly, C. Gerard, J. M. Drazen, and J. Stamler. 1994. Constitutive and inducible nitric oxide synthase gene expession, regulation and activity in human lung epithelial cells. Proc. Natl. Acad. Sci. USA 91: 10089-10093 [Abstract/Free Full Text].

9. Furchgott, R. F.. 1983. Role of endothelium in responses of vascular smooth muscle. Circulation Res. 53: 557-573 [Free Full Text].

10. Werner-Felmayer, G., E. R. Werner, D. Fuchs, A. Hausen, G. Reibnegger, and H. Wachter. 1991. On multiple forms of NO synthase and their occurances in human cells. Res. Immunol. 142: 555-600 [Medline].

11. Stuehr, D. J., and M. A. Marletta. 1987. Synthesis of nitrite and nitrate in murine macrophage cell lines. Cancer Res. 47: 5590-5594 [Abstract/Free Full Text].

12. Marletta, M. A., P. S. Yoon, R. Iyengar, C. P. Leaf, and J. S. Wishnot. 1988. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochem. 27: 8706-8711 . [Medline]

13. Hamid, Q., D. R. Springall, V. Riveros-Moreno, P. Chanez, P. Howarth, A. Redington, J. Bousquet, P. Godard, S. Holgate, and J. M. Polak. 1993. Induction of nitric oxide synthase in asthma. Lancet 342: 1510-1513 [Medline].

14. Alving, K., E. Weitzberg, and J. M. Lundberg. 1990. Increased amount of nitric oxide in exhaled air of asthmatics. Eur. Respir. J. 6: 1368-1371 .

15. Kharitonov, S. A., D. Yates, R. A. Robbins, R. Logan-Sinclair, E. A. Shinebourne, and P. J. Barnes. 1994. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 343: 133-134 [Medline].

16. Yan, Z.-Q., G. K. Hansson, B.-E. Skoogh, and J. O. Lötvall. 1995. Induction of nitric oxide synthase in a model of allergic occupational asthma. Allergy 50: 760-764 [Medline].

17. Persson, M. G., and L. E. Gustafsson. 1993. Allergen-induced airway obstruction in guinea pigs is associated with changes in nitric oxide levels in exhaled air. Acta Physiol. Scand. 149: 461-466 [Medline].

18. Durham, S. R., and A. B. Kay. 1985. Eosinophils, bronchial hyperreactivity and late-phase asthmatic reactions. Clin. Allergy 15: 411-481 [Medline].

19. Gleich, G. J.. 1986. The role of the eosinophil leukocyte in bronchial asthma. Bull. Eur. Physiopathol. Respir. 22: 62-69 .

20. Bousquet, J., P. Chanez, J. Y. Lacoste, G. Barnéon, N. Ghavanian, I. Enander, P. Venge, S. Ahlstedt, J. Simony-Lafontaine, P. Godard, and F.-B. Michel. 1990. Eosinophilic inflammation in asthma. N. Engl. J. Med. 323: 1033-1039 [Abstract].

21. Kung, T. T., H. Jones, G. K. Adams III, S. P. Umland, W. Kreutner, R. W. Egan, R. W. Chapman, and A. S. Watnick. 1994. Characterization of a murine model of allergic pulmonary inflammation. Int. Arch. Allergy Immunol. 105: 83-90 [Medline].

22. Kung, T. T., D. M. Stelts, J. A. Zurcher, H. Jones, S. P. Umland, R. W. Egan, W. Kreutner, and R. W. Chapman. 1995. Interferon-gamma and antibodies to interleukin-5 and interleukin 4 inhibit the pulmonary eosinophilia in allergic mice. Inflam. Res. 44(Suppl. 2):S185-S186.

23. Kung, T. T., D. M. Stelts, J. A. Zurcher, G. K. Adams III, R. W. Egan, W. Kreutner, A. S. Watnick, H. Jones, and R. W. Chapman. 1995. Involvement of IL-5 in a murine model of allergic pulmonary inflammation: prophylactic and therapeutic effect of an anti-IL-5 antibody. Am. J. Respir. Cell Mol. Biol. 13: 360-365 [Abstract].

24. Bradford, M. M.. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248-254 [Medline].

25. Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Rannenbaum. 1982. Analysis of nitrate, nitrite, and (15N) nitrate in biological fluids. Anal. Biochem. 126: 131-138 [Medline].

26. Garlisi, C. G., A. Falcone, T. T. Kung, D. Stelts, K. J. Pennline, A. J. Beavis, S. R. Smith, R. W. Egan, and S. P. Umland. 1995. T cells are necessary for Th2 cytokine production and eosinophil accumulation in airways of antigen-challenged allergic mice. Clin. Immunol. Immunopathol. 75: 75-83 [Medline].

27. Renz, H., H. R. Smith, J. E. Henson, B. S. Ray, C. G. Irvin, and E. W. Gelfand. 1992. Aerosolized antigen exposure without adjuvant causes increased IgE production and increased airway responsiveness in the mouse. J. Allergy Clin. Immunol. 89: 1127-1138 [Medline].

28. Hessel, E. M., A. J. M. Van Oosterhout, C. L. Hofstra, J. J. DeBie, J. Garssen, H. V. Loveren, A. K. C. P. Verheyen, H. F. J. Savelkoul, and F. P. Nijkamp. 1995. Bronchoconstriction and airway hyperresponsiveness after ovalbumin inhalation in sensitized mice. Eur. J. Pharmacol. 293: 401-412 [Medline].

29. Moore, W. M., R. K. Webber, G. M. Jerome, F. S. Tjoeng, T. P. Misko, and M. G. Currie. 1994. L-N6-(1-Iminoethyl) lysine: a selective inhibitor of inducible nitric oxide synthase. J. Med. Chem. 37: 3886-3888 [Medline].

30. Mehta, S., C. M. Lilly, K. H. Haley, K. Asano, A. F. Massaro, and J. M. Drazen. 1996. Increased nitric oxide production during acute allergic bronchoconstriction. Am. J. Respir. Crit. Care Med. 153: A796 . (Abstr.) .

31. Tamaoki, J., A. Chiyotani, M. Kondo, and K. Konno. 1995. Role of NO generation in beta -adrenoceptor-mediated stimulation of rabbit airway ciliary motility. Am. J. Physiol. 268: C1342-C1347 [Abstract/Free Full Text].

32. Barnes, P. J., and M. G. Belvisi. 1993. Nitric oxide and lung disease. Thorax 48: 1034-1043 [Medline].

33. Rees, D. D., R. M. J. Palmer, R. Schulz, M. F. Hodson, and S. Moncada. 1990. Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo. Br. J. Pharmacol. 101: 746-752 [Medline].

34. Misko, T. P., W. M. Moore, T. P. Kasten, G. A. Nickols, J. A. Corbett, R. G. Tilton, M. L. McDaniel, J. R. Williamson, and H. G. Currie. 1993. Selective inhibition of inducible nitric oxide synthase by aminoguanidine. Eur. J. Pharmacol. 233: 119-125 [Medline].

35. Tracey, R. W., J. Tse, and G. Carter. 1995. Lipopolysaccharide-induced changes in plasma nitrite and nitrate concentrations in rats and mice: pharmacological evaluation of nitric oxide synthase inhibitors. J. Pharmacol. Exp. Ther. 272: 1011-1015 [Abstract/Free Full Text].

36. Laszlo, F., S. M. Evans, and B. J. R. Whittle. 1995. Aminoguanidine inhibits both constitutive and inducible nitric oxide synthase isoforms in rat intestinal microvascular in vivo. Eur. J. Pharmacol. 272: 169-175 [Medline].

37. Salvemini, D., P. T. Manning, B. S. Zweifel, K. Seibert, J. Connor, M. G. Currie, P. Needleman, and J. L. Masferrer. 1995. Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors. J. Clin. Invest. 96: 301-308 .

38. Nathan, C.. 1992. Nitric oxide as a secretory product of mammalian cells. FASEB J. 6: 3051-3064 [Abstract].

39. Moncada, S., R. M. J. Palmer, and E. A. Higgs. 1991. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev. 43: 109-141 [Medline].

40. Shaul, R. W., A. J. North, L. C. Wu, L. B. Wells, T. S. Brannon, K. S. Lau, T. Michel, L. R. Margraf, and R. A. Star. 1994. Endothelial nitric oxide synthase is expressed in cultured human bronchial epithelium. J. Clin. Invest. 94: 2231-2236 .

41. Robbins, R. A., P. J. Barnes, D. R. Springall, J. B. Warren, O. J. Kwan, L. D. K. Buttery, A. J. Wilson, D. A. Geller, and J. M. Polak. 1994. Expression of inducible nitric oxide in human lung epithelial cells. Biochem. Biophys. Res. Commun. 203: 209-218 [Medline].

42. Feder, L. S., D. Manfra, T. H. Paster, X. Fernandez, T. T. Kung, D. Stelts, and W. Kreutner. 1996. Lipopolysaccharide (LPS) but not antigen sensitization and challenge of mice alters iNOS expression in the lung. Am. J. Respir. Crit. Care Med. 153: A289 . (Abstr.) .

43. Kung, T. T., D. Stelts, J. Zurcher, H. Jones, S. P. Umland, W. Kreutner, R. W. Egan, and R. W. Chapman. 1995. Mast cells modulate allergic pulmonary eosinophilia in mice. Am. J. Respir. Cell Mol. Biol. 12: 404-409 [Abstract].

44. Wardlaw, A. J., S. Dunnette, G. J. Gleich, J. V. Collins, and A. B. Kay. 1988. Eosinophils and mast cells in bronchoalveolar lavage in subjects with mild asthma. Am. Rev. Respir. Dis. 173: 62-69 .

45. Ignarro, L. J., R. E. Byrns, G. M. Buga, and K. S. Wood. 1987. Endothelium-derived relaxing factor from pulmonary artery and vein possesses pharmacologic and chemical properties identical to those of nitric oxide radical. Circ. Res. 61: 866-879 [Abstract/Free Full Text].

46. Kuo, H.-P., S. Liu, and P. J. Barnes. 1992. The effect of endogenous nitric oxide on neurogenic plasma exudation in guinea pig airways. Eur. J. Pharmacol. 221: 385-388 [Medline].

47. Belenky, S. N., R. A. Robbins, S. I. Rennard, G. L. Gossman, K. J. Nelson, and I. Rubinstein. 1993. Inhibitors of nitric oxide synthase attenuate human neutrophil chemotaxis in vitro. J. Lab. Clin. Med. 122: 388-394 [Medline].

48. Belenky, S. N., R. A. Robbins, and I. Rubinstein. 1993. Nitric oxide synthase inhibitors attenuate human monocyte chemotaxis in vitro. J. Leuk. Biol. 53: 498-503 [Abstract].

49. Robbins, R. A., F. A. Romero, K. J. Nelson, G. E. Hill, and R. G. Townley. 1995. Inhibitors of nitric oxide attenuate human eosinophil chemotaxis. Am. J. Respir. Crit. Care Med. 151: A238 . (Abstr.) .

50. Taylor-Robinson, A. W., F. Y. Liew, A. Severn, D. Xu, S. J. McSorley, P. Garside, J. Dardon, and R. S. Phillips. 1994. Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells. Eur. J. Immunol. 24: 980-984 [Medline].

51. Gajewski, T. F., and F. W. Fitch. 1988. Anti-proliferative effect of IFN-gamma in immune regulation. I. IFN-gamma inhibits the proliferation of TH2 but not TH1 murine helper T lymphocyte clones. J. Immunol. 140: 4245-4252 [Abstract].

52. Barnes, P. J., and F. Y. Liew. 1995. Nitric oxide and asthmatic inflammation. Immunol. Today 16: 128-130 [Medline].

53. Mosmann, T. R., and R. L. Coffman. 1989. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Ann. Rev. Immunol. 7: 145-173 [Medline].




This article has been cited by other articles:


Home page
 Toxicol SciHome page
C. C. Dong, X. J. Yin, J. Y. C. Ma, L. Millecchia, M. W. Barger, J. R. Roberts, X.-D. Zhang, J. M. Antonini, and J. K. H. Ma
Exposure of Brown Norway Rats to Diesel Exhaust Particles Prior to Ovalbumin (OVA) Sensitization Elicits IgE Adjuvant Activity but Attenuates OVA-Induced Airway Inflammation
Toxicol. Sci., November 1, 2005; 88(1): 150 - 160.
[Abstract] [Full Text] [PDF]

Home page
 Toxicol SciHome page
C. C. Dong, X. J. Yin, J. Y. C. Ma, L. Millecchia, Z.-X. Wu, M. W. Barger, J. R. Roberts, J. M. Antonini, R. D. Dey, and J. K. H. Ma
Effect of Diesel Exhaust Particles on Allergic Reactions and Airway Responsiveness in Ovalbumin-Sensitized Brown Norway Rats
Toxicol. Sci., November 1, 2005; 88(1): 202 - 212.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
C. M. Prado, E. A. Leick-Maldonado, D. I. Kasahara, V. L. Capelozzi, M. A. Martins, and I. F. L. C. Tiberio
Effects of acute and chronic nitric oxide inhibition in an experimental model of chronic pulmonary allergic inflammation in guinea pigs
Am J Physiol Lung Cell Mol Physiol, October 1, 2005; 289(4): L677 - L683.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
R. Morita, T. Uchiyama, and T. Hori
Nitric Oxide Inhibits IFN-{alpha} Production of Human Plasmacytoid Dendritic Cells Partly via a Guanosine 3',5'-Cyclic Monophosphate-Dependent Pathway
J. Immunol., July 15, 2005; 175(2): 806 - 812.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
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]

Home page
 J. Immunol.Home page
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]

Home page
 Am. J. Respir. Crit. Care Med.Home page
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]

Home page
 J. Pharmacol. Exp. Ther.Home page
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]

Home page
 J. Pharmacol. Exp. Ther.Home page
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]

Home page
 Eur Respir JHome page
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]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
M. Feletou, M. Lonchampt, F. Coge, J.-P. Galizzi, C. Bassoullet, C. Merial, P. Robineau, J. A. Boutin, P. L. Huang, P. M. Vanhoutte, et al.
Regulation of murine airway responsiveness by endothelial nitric oxide synthase
Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L258 - L267.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
H. Kobayashi, R. Hataishi, H. Mitsufuji, M. Tanaka, M. Jacobson, T. Tomita, W. M. Zapol, and R. C. Jones
Antiinflammatory Properties of Inducible Nitric Oxide Synthase in Acute Hyperoxic Lung Injury
Am. J. Respir. Cell Mol. Biol., April 1, 2001; 24(4): 390 - 397.
[Abstract] [Full Text]

Home page
 Am. J. Respir. Crit. Care Med.Home page
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]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
K. A. Nath, V. Shah, J. J. Haggard, A. J. Croatt, L. A. Smith, R. P. Hebbel, and Z. S. Katusic
Mechanisms of vascular instability in a transgenic mouse model of sickle cell disease
Am J Physiol Regulatory Integrative Comp Physiol, December 1, 2000; 279(6): R1949 - R1955.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
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]

Home page
 Am. J. Respir. Crit. Care Med.Home page
H. SUGIURA, M. ICHINOSE, T. OYAKE, Y. MASHITO, Y. OHUCHI, N. ENDOH, M. MIURA, S. YAMAGATA, A. KOARAI, T. AKAIKE, et al.
Role of Peroxynitrite in Airway Microvascular Hyperpermeability during Late Allergic Phase in Guinea Pigs
Am. J. Respir. Crit. Care Med., August 1, 1999; 160(2): 663 - 671.
[Abstract] [Full Text]

Home page
 Pharmacol. Rev.Home page
M. A. Giembycz and M. A. Lindsay
Pharmacology of the Eosinophil
Pharmacol. Rev., June 1, 1999; 51(2): 213 - 340.
[Abstract] [Full Text] [PDF]

Home page
 J. Exp. Med.Home page
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 page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
W. S. Cruz, J. A. Corbett, W. J. Longmore, and M. A. Moxley
Nitric oxide participates in early events associated with NNMU-induced acute lung injury in rats
Am J Physiol Lung Cell Mol Physiol, February 1, 1999; 276(2): L263 - L268.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Xiong, G. Karupiah, S. P. Hogan, P. S. Foster, and A. J. Ramsay
Inhibition of Allergic Airway Inflammation in Mice Lacking Nitric Oxide Synthase 2
J. Immunol., January 1, 1999; 162(1): 445 - 452.
[Abstract] [Full Text] [PDF]