Introduction

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Nitric oxide (NO) has been described as a potent intracellular mediator produced by, and acting upon, many cells of the body (1, 2). Recent studies have suggested that NO may be involved in asthma. Inducible nitric oxide synthase (iNOS) is constitutively expressed in normal bronchial epithelial cells, with increased levels in epithelial cells from patients with asthma (3). Patients with asthma also have significantly higher levels of exhaled NO than do normal individuals (4). These studies suggest that in asthma, NO is upregulated, and that the epithelial cell is a prime source of NO.

The role of the alveolar macrophage (AM) in the pathogenesis of asthma has not been well defined. AM are the predominant leukocytes found in the air space under homeostatic conditions, and most importantly, the AM has numerous regulatory characteristics (8). Macrophages have the ability to make cytokines in response to both nonspecific stimuli, such as endotoxin, and specific antigen stimulation via IgE-mediated pathways (8, 9). Bronchoalveolar lavage fluid (BALF) from asthmatic individuals contains high levels of a number of inflammatory cytokines produced by AM, including tumor necrosis factor (TNF), interleukin-1 (IL-1), and macrophage inflammatory protein- (MIP-1) (10). Levels of TNF and IL-1 are increased in numerous inflammatory conditions and have prominent effects on airway epithelial cells, which include the induction of other cytokines and inflammatory mediators (14, 15). MIP-1 has been shown to have chemoattractant activity for a number of cell populations associated with exacerbations of asthma, including T lymphocytes, eosinophils, and basophils (16). Although considerable attention has been devoted to the regulation of NO by inflammatory cytokines, and also to the role of NO as an important effector molecule in immune function, very little information has been reported about the role of NO in modulating human AM activities (17). The purpose of the present study was to investigate the effect of NO on cytokine production by human AM.

	Materials and Methods

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Preparation of Macrophages

AM were obtained by fiberoptic bronchoscopy from normal volunteers as previously described (18). All volunteers provided written informed consent, which was approved by the Institutional Review Board of the Cleveland Clinic. The tip of the bronchoscope was wedged into the right middle lobe or the lingula. A total of 300 ml of saline was instilled by gravity in 60-ml aliquots, and was withdrawn by gentle aspiration. Lavage fluid was filtered and the cells washed with Hanks' balanced salt solution (HBSS) (GIBCO, Grand Island, NY). Cell number was determined with a hemocytometer, and differential cell counts were performed with a modified Wright's stain (Hema-3 stain; Biochemical Sciences, Inc., Bridgeport, NJ). The average cell yield from 14 normal volunteers was (23 ± 14) × 10⁶ (mean ± SD) with 97 ± 2% AM. The normal volunteers included 12 nonsmokers and two smokers. Results for smokers and nonsmokers were combined because the means of results for the two groups did not differ. Cells were resuspended in RPMI 1640 or Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% human AB serum (Gemini, Calabasas, CA), L-glutamine, and antibiotics. Macrophages were plated at 3 × 10⁵ cells per well in 24-well culture plates, and were allowed to adhere for 1 h at 37°C in a moist, 5% CO₂ incubator. Nonadherent cells were removed by washing with warmed RPMI. The adherent cell population comprised > 99% AM.

Reagents and Drugs

Salmonella typhimurium lipopolysaccharide (LPS) was obtained from Sigma Chemical Co. (St. Louis, MO) and was used at 0.5 µg/ml for all experiments. 2,2-(hydroxynitrosohydrazono)-bis-ethanamine (DETA NONOate) was obtained from Cayman Chemical Company (Ann Arbor, MI) and used at indicated concentrations. DETA NONOate releases nitric oxide in culture, with t_1/2 = 20 h at 37°C (19).

Cytokine Assays

Macrophages were incubated for 24 h with LPS with or without DETA NONOate. Cell-free culture supernatants were collected and assayed for cytokines with an enzyme-linked immunosorbent assay (ELISA) (Endogen, Cambridge, MA; Cayman Chemical Company, Ann Arbor, MI; or R&D Systems, Minneapolis, MN). The sensitivity of the assays ranged from 3 to 31 pg/ml. All cytokine assays were done in duplicate, and the coefficient of variation (CV) for all assays was < 10%.

Measurement of NO Production by Chemiluminescence

The NO production in culture was measured with a nitric oxide analyzer (NOA; Siever, Boulder, CO). Because RPMI 1640 contains Ca(NO₃)₂, DMEM was used for these experiments. Comparison experiments demonstrated similar results with both media (data not shown). Nitrate and nitrite present in culture supernatants (4 µl) were converted to NO with a saturated solution of VCl₃ in 0.8 M HCl, and the NO was detected through a gas-phase chemiluminescent reaction between NO and ozone. Nitrite and nitrate standards were also tested. The nitrite and nitrate standards displayed linearity between 10 nM and 125 µM (r²  0.995 for all experiments), and the NO they released was detected with equal efficiency (< 15% difference in detection at any concentration). NO levels were determined by interpolation from known standard curves.

MTT Assay

The viability of AM after various treatments was quantified with the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenol tetrazolium bromide (MTT) cleavage assay (Boehringer Mannheim, Indianapolis, IN). The amount of MTT reduced to its purple formazan derivative by viable cells was quantified spectrophotometrically at 540 nm. There is a linear relationship between the formazan generated and the number of viable cells present (20).

Statistical Analysis

The results of experiments were analyzed for their statistical significance with Wilcoxon's signed ranks test, using GraphPad Prism software (GraphPad Software, Inc., San Diego, CA). A value of P < 0.05 was considered statistically significant.

	Results

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NO Production from DETA NONOate

Culturing AM in the presence of DETA NONOate results in dose-dependent exposure to NO as measured by the amount of nitrate and nitrite present in the culture medium as determined by chemiluminescence (Figure 1). Endogenous production of NO was not detected for AM cultures without DETA NONOate and incubated in medium with or without LPS.

View larger version (18K): [in this window] [in a new window]   Figure 1.   NO production by DETA NONOate in AM cultures with or without LPS after 24 h incubation at 37°C. NO production was measured with an NO analyzer. Data represent the mean ± SEM of four or five experiments.

Effect of NO on Inflammatory Cytokine Production

To evaluate the effect of NO on stimulated AM, DETA NONOate was added to LPS-stimulated AM. DETA NONOate significantly (P < 0.03) suppressed TNF and IL-1 secretion in a dose-dependent manner (Figure 2). The secretion of MIP-1 was also suppressed by DETA NONOate (Figure 3). Exposure of unstimulated AM to DETA NONOate for 24 h did not affect the low basal levels of TNF and IL-1 (Table 1).

View larger version (17K): [in this window] [in a new window]   Figure 2.   Dose dependence of NO inhibition of alveolar macrophage TNF and IL-1 secretion. AM were stimulated with LPS and incubated with or without DETA NONOate for 24 h (n = 10 experiments). Secretion of all cytokines by LPS-stimulated macrophages was significantly (P  0.03; Wilcoxon's signed ranks test) decreased by 0.1 to 1 mM DETA NONOate.

View larger version (16K): [in this window] [in a new window]   Figure 3.   Dose dependence of NO inhibition of AM secretion of MIP-1. AM were stimulated with LPS and incubated with or without DETA NONOate for 24 h. Data represent the mean ± SEM of three experiments.

To assess whether NO simply blocked cytokine release, levels of both cell-associated and secreted IL-1 from LPS-stimulated AM were measured (Figure 4). Both cell-associated and secreted IL-1 were suppressed to a similar extent.

View larger version (22K): [in this window] [in a new window]   Figure 4.   Comparison of the effect of DETA NONOate on cell-associated (A) and secreted (B) IL-1. Data represent the mean ± SEM of three experiments.

Effect of NO on AM Viability

To determine whether the observed decrease in cytokine secretion resulted from cytotoxic effects of NO, we studied the effects of LPS and DETA NONOate on the viability of the macrophages. AM were cultured for 24 h in the presence of various concentrations of DETA NONOate with or without LPS, and their mitochondrial respiratory activity was subsequently measured with the MTT assay. As depicted in Figure 5, no significant differences were observed in the mitochondrial activity of macrophages cultured in various concentrations of DETA NONOate with or without LPS.

View larger version (22K): [in this window] [in a new window]   Figure 5.   Effect of DETA NONOate on the viability of AM. AM were cultured with indicated concentrations of DETA NONOate (A) and LPS + DETA NONOate (B). After 24-h culture, mitochondrial respiratory activity was determined with the MTT assay. Data represent mean ± SEM of four experiments performed in triplicate.

	Discussion

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The role of NO in regulating cytokine production by human AM has not previously been explored. In the present study, adherent populations of normal human AM, both unstimulated and LPS-stimulated, were exposed to NO generated from DETA NONOate. NO inhibited LPS-stimulated inflammatory cytokine production (TNF, IL-1, MIP-1) by these AM. NO did not affect basal cytokine levels. As previously reported by Feelisch and Stamler (17), endogenous production of NO by unstimulated or LPS-stimulated human AM was not detected. Studies of mitochondrial activity (MTT) indicated that NO was not cytotoxic for unstimulated or LPS-stimulated AM. These findings indicate that NO is a potent inhibitor of cytokine production by stimulated human AM.

The concentration of NO to which AM are exposed in vivo is difficult to determine. NO has not been detected in airway lining fluid in solution or in complexes with transition metals (21). However, the studies of Gaston and colleagues (21) suggest that NO exists in the lung in the form of metabolic intermediaries that serve to modulate the bioactivity and toxicity of NO. Gaston and colleagues found nitrite (10 to 20 µM) and S-nitrosothiols in lung lining fluid, and both were increased (~ 100 µM nitrite) after 10 min of inhalation in patients receiving 40 ppm of NO gas for pulmonary hypertension. In our studies, the concentration of NO (nitrite and nitrate) released into the medium by DETA NONOate and shown to inhibit cytokine secretion by AM was of the same order of magnitude (~ 200 to ~ 800 µM) as that observed in the patients given exogenous NO.

In contrast to human AM, rat AM produce NO upon LPS stimulation. When NO production by rat macrophages is blocked by the L-arginine analogue N^G-monomethyl-L-arginine (NMMA), these cells demonstrate an increase in IL-1 and IL-6 secretion (22). Furthermore, the NO donor S-nitroso-N-acetyl-D, L-penicillamine (SNAP) induced dose-dependent inhibition of IL-1 production in LPS-stimulated rat AM in which endogenous NO production was blocked. In contrast to our finding that the inhibitory effects of NO in human AM did not appear to be cytokine specific, no increase in TNF was noted with blocking of endogenous NO production in rat AM. The reason for the apparent cytokine specificity of NO in the rat is unknown, but may reflect a species difference in regulatory mechanisms.

Recently, NO inhalation has been used to improve arterial blood oxygenation in patients with adult respiratory distress syndrome (23). High levels of IL-8 and IL-6 decreased in these patients' BALF after NO inhalation. In a control group of ARDS patients not treated with NO, the IL-8 and IL-6 levels in BAL were not significantly changed. These in vivo results support our in vitro observations that NO inhibits inflammatory-cytokine production.

The release of macrophage proinflammatory cytokines is generally secondary to increased gene transcription, which is controlled by activation of transcription factors such as nuclear factor-B (NF-B) (24). Interestingly, NO has been shown in human endothelial cells to inhibit the activation of NF-B by inducing and stabilizing IB (25, 26). Whether such a mechanism is operative in human AM requires further study.

We have shown that NO functions in an antiinflammatory capacity through downregulation of proinflammatory-cytokine secretion by normal human AM. Whether AM from patients with inflammatory diseases such as asthma are subject to such regulation has not been investigated. However, for the patient with asthma, the upregulation of NO production in the lung, as suggested by increased iNOS expression and exhaled NO, may be a mechanism for maintaining pulmonary homeostasis by decreasing inflammatory-cytokine production by AM.