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, N^G-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- (IFN-) 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- (50). By contrast, Th2 cells neither produce nor are affected by NO (50). IFN- is known to inhibit Th2 cell proliferation (51). Thus, production of large amounts of NO could reduce the secretion of IFN- 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- 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.