Nasal Polyp Derived Superoxide Anion Dose-Dependent Inhibition by Nitric Oxide and Pathophysiological Implications

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1 Nasal Polyp Derived Superoxide Anion Dose-Dependent Inhibition by Nitric Oxide and Pathophysiological Implications MERITXELL PASTO, ELIE SERRANO, EMANUELLE UROCOSTE, MARIE-ALINE BARBACANNE, ANNIE GUISSANI, ALAIN DIDIER, MARIE-BERNADETTE DELISLE, JACQUES RAMI, and JEAN-FRANÇOIS ARNAL Services d Exploration Fonctionnelle Respiratoire et INSERM U397, de Pneumologie et d Allergologie, d ORL, et d Anatomopathologie, Rangueil, France; Laboratoire de Synthèse, Physico-Chimie et Radiobiologie, Université Paul Sabatier, Toulouse Cedex, France, and Institut Curie, Bat 112, INSERM U350, Centre Universitaire, Orsay, France The epithelium of the paranasal sinuses produces nitric oxide (NO), which probably plays a major role in the nonspecific defense of these cavities through its bacteriostatic and cilia motility stimulation properties. Abundant eosinophils of nasal polyps potentially generate superoxide anion ( ), but NO and inactivate reciprocally. The purpose of the present work was to evaluate the relationship between NO concentrations and nasal polyp production of. Polyp fragments from 24 patients were studied using histological examination and lucigenin-enhanced chemiluminescence (to assess production). The effect of various concentrations of exogenous NO on chemiluminescent signals was assessed. Basal and phorbol ester-stimulated production varied largely among patients, but both were highly related to eosinophilic infiltration. A slow releasing NO donor DETA NONOate (DETA/NO NOC-18) dose dependently inhibited lucigenin-enhanced chemiluminescence from phorbol ester-stimulated polyp fragments, with an EC 50 of 1.5 mm. The NO concentration in normal maxillary sinus was estimated about 10 ppm (i.e., 0.5 M in aqueous phase) (Lundberg, et al. Nature Med 1995;1:370). Calculations revealed that the DETA NONOate 0.75 mm and 1.5 mm generate steady-state concentrations of NO of 0.5 M and 2.5 M, respectively. In conclusion, the NO concentration present in paranasal sinuses appears to partially suppress (approximately 20 40%) production from polyp eosinophils. Conversely, phagocyticderived could contribute to decrease sinus NO concentration, further altering this natural local defense. Together, these events could participate in chronic inflammation and contribute to the pathophysiology of nasal polyps. Nitric oxide (NO), a free radical gas previously considered merely an atmospheric pollutant, is now recognized to be one of the key mediators in many physiological and pathological processes. NO is generated from arginine by a family of enzymes, the NO synthases, and acts as an autocrine and paracrine messenger (1 3). NO also plays a major role in nonspecific host defense due to its cytotoxicity toward tumor cells and microorganisms. Inducible NO synthase (type II NO synthase) can be expressed after induction by proinflammatory cytokines or by bacterial lipopolysaccharide in many cell types (4). Thus, NO appears as a second line of defense against hard to kill pathogens in most mammalian tissues (4). The first line of nonspecific host defense is the NADPH-oxidase of the phagocytic cells (monocyte macrophage and polymorphonuclears), (Received in original form February 29, 2000 and in revised form June 28, 2000) This work was supported in part by the Délégation à la Recherche Clinique des Hôpitaux de Toulouse (97-51-H) and by INSERM. Correspondence and requests for reprints should be addressed to J.-F. Arnal, Service d exploration fonctionnelle respiratoire, CHU Rangueil, Toulouse Cedex, France. Am J Respir Crit Care Med Vol 163. pp , 2001 Internet address: which, upon activation, generate large amounts of superoxide anion ( ) and derived reactive oxygen species (ROS, as hydrogen peroxide and hydroxyl radical) (5, 6). However, this schematic view probably does not apply to all sites of the organism. The epithelia of the respiratory tract, bronchial (7) as well as paranasal sinuses (8), constitutively express the inducible NO synthase. As a consequence of the low air renewal in paranasal sinuses, very high concentrations of NO gas (5 20 parts per million [ppm]) have been measured in these cavities (8). Thus, NO could play a critical role in the physiology and pathology of the upper respiratory tract, because in addition to its role in nonspecific host defense (9), NO also stimulates ciliary motility (10). In the paranasal sinuses, NO appears to represent the first line of host defense, contributing to the sterility of these cavities (11). Inflammation of the sinus (i.e., sinusitis) induces the recruitement of phagocytic cells, which, in this particular case, represent a second line of defense (11). Thus, the strategy for host defense in the paranasal sinuses appears to be the reverse of that encountered in other cavities (such as the pulmonary alveola and peritoneal cavity) where resident macrophages are constitutively present to kill pathogens, whereas basal levels of NO are very low (12). Because NO and both contain an unpaired electron, they rapidly react together, leading to their reciprocal inactivation and eventually to peroxynitrite (ONOO ) (13, 14). Peroxynitrite can, in turn, elicit protein tyrosine nitration, although other mechanisms, such as oxidation of nitrites by myeloperoxidase, can also elicit similar protein alterations (15). The in vivo interaction between NO and, as well as the consequences for host defense and cell toxicity, are hard to predict for the following reasons: (1) in vivo concentrations of NO cannot be easily determined with the technologies currently available, and the estimates in the literature vary widely according to the authors, (2) the generation of is even more difficult to determine, due to the very short half-life of this ROS. Moreover, the validity of the most sensitive and commonly employed technique, lucigenin-enhanced chemiluminescence, has recently been questioned (16 18), and (3) the very rapid interaction between NO and further complicates the assessment of each radical species (13, 14). In the present study, we first applied electron spin resonance (ESR) spectroscopy using DMPO as spin trap, ferricytochrome c reduction, and lucigenin-enhanced chemiluminescence to assess the production of a macrophage cell line RAW and validate the latter technique. We also compared the effect of three NO donors on ESR and chemiluminescent signals. Fragments of nasal polyps from patients who underwent surgery for nasal polyposis were then studied by histological examination and lucigenin-enhanced chemiluminescence. The effect of various concentrations of an NO donor, DETA NONOate, on chemiluminescent signals from na-

2 146 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL sal polyps fragments was therefore investigated, and the NO concentrations generated by DETA NONOate were calculated and compared with those reported in maxillary paranasal sinuses. The final goal of this work was to determine to what extent the concentration of NO present in paranasal sinuses inactivates the production by phagocytes. METHODS Cell Culture and Materials A mouse macrophage cell line (RAW 264.7; American Type Culture Collection, Rockville, MD) was cultured in Dulbecco s modified Eagle medium with 10% heat-inactivated fetal calf serum (GIBCO) supplemented with gentamycin (0.1 mg/ml) and amphotericin B (125 ng/ml) in a 5% C -containing atmosphere. RAW were used when cells approached confluency (100,000/cm 2 ) in the flasks or in 96 microwells. Except when specified, all reagents were purchased from Sigma (St. Louis, MO): superoxide dismutase (SOD, ref. S-2515; phorbol 12-myristate 13-acetate (PMA), identical to 12-O-tetradecanoylphorbol 13-acetate [TPA]), catalase (ref. C-40), 5,5-dimethyl-1 pyrroline-n-oxide (DMPO), diethylenetriaminepentaacetic acid (DTPA, ref. D-751), and activated charcoal. DETA/NO NOC-18 and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) were purchased from Alexis Biochemicals (San Diego, CA). DMPO was purified before use by treatment with activated charcoal for 15 min as reported in the literature (19), passed through a membrane filter (0.2 m; Sartorius, Göttingen, Germany), aliquoted, protected from light and stored frozen at 20 C. Patients with Nasal Polyposis The procedures employed in this study were reviewed and approved by the local Ethics Committee (Comité consultatif de protection des personnes). The study was performed in 24 patients with nasal polyposis. The diagnosis of patients with nasal polyposis was based on the endoscopic observation of nasal polyps. Patients characteristics were mean age yr and sex ratio 16 males 8 females. Clinical symptoms were assessed the day of the NO measurement. The presence of symptoms (nasal obstruction, rhinorrhoea, and sneezing) the day of the NO measurement was noted, and the intensity (0, 1, 2 or 3) of each symptom was evaluated. The symptom scores were nasal obstruction , rhinorrhea , and sneezing All patients underwent surgery for nasal polyposis, and the nasal polyps were removed surgically and stored at 4 C in Dulbecco s modified Eagle medium supplemented with gentamycin (0.1 mg/ml) and amphotericin B (125 ng/ml) for less than 1 h before being processed in the laboratory. The nasal polyps were then cut with a razor blade into fragments weighing 10 mg on average. A nasal polyp from each patient was cut into two large fragments, one for histological examination, the other being segmented, depending on its size, into 15 to 25 fragments of approximately 10 mg. Assessment of O 2 Production ESR measurements. RAW were cultured in a 25-cm 2 flask, washed with phosphate-buffered saline (PBS), and then incubated with a mix containing 150 mm DMPO, 1 g/l glucose, 0.2 g/l CaCl 2, g/l DTPA, 0.15 g/l NaCl, and 0.37 g/l KCl in sodium phosphate buffer (2.35 g/l NaH 2 PO 4 /7.61 g/l Na 2 HPO 4, ph 7.4) containing phorbol myristate acetate (PMA) 1 M for 10 min. The entire procedure was performed at 37 C. The supernatant was then transferred to a flat quartz cell that was inserted in a TM 110 Bruker cavity. ESR spectra were recorded at room temperature with an ER 200 D Bruker spectrometer by starting a 3 min scan 5 min after the end of the incubation with the cells. The ESR spectrometer operated at 9.66 GHz with high frequency at 100 khz, modulation amplitude 1 G, time constant 0.5 s, microwave power 10 mw, field: midrange at 3,500 G, and scan range 20 G/min. The intensity of the ESR signal was calculated by adding the height of the four peaks, and expressed in arbitrary units as previously described (20). In some experiments, 5 min after the addition of a NO donor, RAW were stimulated with 1 M PMA. SOD inhibitable reduction of cytochrome c. O 2 production was measured as the SOD inhibitable reduction of cytochrome c (21). RAW (400,000 cells/well) were preincubated with RPMI for 15 min at 37 C, washed once with RPMI, and incubated with 1 ml RPMI containing 1 mg/ml cytochrome c with or without SOD (200 IU/ml) in humidified air on a shaking table. Cytochrome c reduction was determined at zero time to obtain basal values and after the indicated time, the absorbance of the medium was read spectrophotometrically at 550 nm against a distilled water blank. Reduction of cytochrome c in the presence of SOD was subtracted from the values without SOD: the proportion of superoxide specific reduction of cytochrome c (i.e., SOD inhibitable) was between 30 and 50% depending on the experiments. In some experiments, RAW were stimulated with 1 M PMA, 5 min after the addition of an NO donor. Lucigenin-enhanced chemiluminescence. RAW were cultured in 96-microwells. The production of reactive oxygen intermediates was measured using the chemiluminogenic probe lucigenin. The medium was removed from the wells and the cells washed twice with Krebs Henseleit bicarbonate (KHB) (composition [mm]: NaCl 99.0, KCl 4.69, CaCl , MgSO 4 1.2, NaHCO 3 25, K 2 HPO , Na- HEPES 20, D-glucose 11.1) with 1 M PMA for 10 min, and lucigenin (250 M final) was then added. The 96 microwells were thermostatically (37 C) controlled, and chemiluminescence was triggered with 1 m PMA through an automatic injector (200 l of final volume in each well), continuously monitored for 30 min and expressed in relative light unit/min. An average production was calculated from that of the different fragments, and this value was then used in correlation with histological scores. In some experiments, RAW were stimulated with 1 m PMA 5 min after the addition of an NO donor. Calculation of the Steady State Concentration of NO Generated by DETA/NO NOC-18 Decomposition of NONOates was monitored spectrophotometrically, at 37 C, in 0.1 M phosphate buffer, ph 7.4. For simulation of NO production, it was considered that NONOates release two equivalents of NO and that NO auto-oxidizes in aqueous solutions according to the following equations (22): with [ ] being constant and equal to 220 M. The second integration method of Runge-Kutta was used (23). A small integration step was used to simulate the reactions at the initial times; as a rule, the initial integration step size was of the order of onehundredth of the reaction time of the fastest reaction. The calculations were performed using the spreadsheet Excel 98 (Microsoft Office). Histological Examination The abundance of inflammatory cells was estimated using a semiquantitative score: 1 for a low number of inflammatory cells, 2 for a moderate infiltration by inflammatory cells on the chorion surface, and 3 for an intense infiltration by inflammatory cells on the chorion surface. The percentage of eosinophils in the inflammatory cells was also estimated using a semiquantitative score: 1 for 10% of eosinophils, 2 for 10 50% of eosinophils, and 3 for 50% of eosinophils. Finally, fibrosis was semiquantified as follows: 1 for chorion s edema, 2 for normal chorion, and 3 for constituted sclerosis of the chorion. Statistical Analysis The data were expressed as mean of triplicate values. The relation between the variables was calculated using Pearson s rank-correlation coefficients. p values of 0.05 were considered significant. Analysis was performed with the SPSS program. RESULTS 4NO H 2 O 4 N + 4H dno [ ] dt = 4k[ NO ] 2 [ ] Validation of O 2 Measurements ESR signals detected using DMPO as spin trap. After 15 min incubation of DMPO with RAW stimulated by 1 m PMA, a typical ESR signal was obtained resulting from the DMPO-OH adduct (Figure 1A). This adduct could result

3 Pasto, Serrano, Urocoste, et al.: NO and Superoxide Interactions in Nasal Polyps 147 Figure 1. Representative tracings of electron spin resonance (ESR) spectra (A) and lucigenin-enhanced chemiluminescence (B) obtained from RAW 264.7, and of lucigenin-enhanced chemiluminescence (C) obtained from nasal polyp. (A) The cells ( ) were incubated in the presence of the spin trap DMPO and stimulated with 1 M 12-O-tetradecanoylphorbol 13-acetate (TPA) for 15 min. (B) The cells ( ) were incubated in the presence of lucigenin. Two right tracings: basal. Two left tracings: stimulation with 1 M TPA, being applied just before the begining of the 15 min recording. (C) A fragment of nasal polyp was incubated in the presence of lucigenin. Two right tracings: basal. Two left tracings: stimulation with 1 M TPA, being applied just before the begining of the 45 min recording. RLU relative light units, mg mg tissue, s second. from hydroxyl radical trapping by DMPO in the extracellular medium. However, it is well known that the DMPO-OOH adduct, resulting from the trapping of, rapidly decomposes into a more stable adduct: DMPO-OH (24). To identify the ROS released by the RAW and initially trapped by DMPO, the effects of SOD and catalase were tested as previously reported (25, 26). When SOD 30 U/ml was coincubated, the ESR signal was completely suppressed (not shown). In contrast, neither denatured SOD (15 min boiling) nor catalase (2,000 U/ml) affected the ESR signal (not shown). Together, these results demonstrate that the ESR adduct DMPO-OH detected in the supernatant of RAW originated from the trapping of extracellular. The ESR signal given by unstimulated (basal) cells was about 10-fold the baseline (Figure 1). As shown in Figure 2, three NO donors (sodium nitroprusside, DETA/NO NOC-18, and SNAP) dose dependently inhibited the amplitude of the ESR adduct DMPO-OH from RAW Cytochrome c reduction technique. RAW stimulated by 1 m PMA induced an immediate SOD-inhibitable cytochrome c reduction, which was suppressed if SOD was previously boiled for 15 min. Catalase (2,000 U/ml) had no effect (not shown). These results demonstrate that SOD-inhibitable cytochrome c reduction signal elicited by PMA-stimulated RAW is mainly due to extracellular. Lucigenin-elicited chemiluminescent signals from macrophage cell line. RAW stimulated by 1 m PMA induced an immediate lucigenin-elicited chemiluminescent signal. Representative tracings are shown in Figure 1B. SOD 100 U/ml and diphenyleneiodonium (DPI) 100 M inhibited 90% of the lucigenin-enhanced chemiluminescent signal. In contrast, coincubation with denatured SOD (15 min boiling) or with catalase (2,000 U/ml) did not affect the morphology or the amplitude of the chemiluminescent signal (not shown). Altogether, these results demonstrate that the lucigenin-enhanced chemiluminescent signal elicited by PMA-stimulated RAW is mainly due to extracellular. As shown in Figure Figure 2. The dose response effect of SNP, DETA/NO NOC-18, and SNAP was studied on the ESR spectra generated (hatched bars) and on the lucigenin-enhanced chemiluminescence (black bars) by TPA-stimulated RAW Data are the averages of triplicate incubations and representative of two different experiments.

4 148 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 3. (A, B) Reproducibility of lucigenin-elicited chemiluminescence (RLU relative light units, mg mg tissue, s second) from the differents fragments of the polyp patients. 2, the three NO donors dose dependently inhibited the amplitude of the lucigenin-enhanced chemiluminescent signal from RAW Histological Examination of Nasal Polyps and Relation to Lucigenin-elicited Chemiluminescent Signals Eight fragments of each polyp were used to study the reproducibility of lucigenin-elicited chemiluminescence. For a given polyp, some variations were found for both basal and TPAstimulated lucigenin-elicited chemiluminescence (Figures 3A and 3B). A mean value was calculated for each polyp and used for subsequent correlations with histology. Large variations in both basal and TPA-stimulated lucigenin-elicited chemiluminescence were also found between patients, and these are ordered from lowest to highest producer in Figures 3A and 3B. Histological examination revealed the presence of various degrees of inflammation as well as fibrosis (Figure 4). Basal lucigenin-elicited chemiluminescence (calculated mean from the eight fragments) was significantly correlated with eosinophils abundance (r 0.37, p 0.05), fibrosis abundance (r 0.3, p 0.05) but not with cell abundance (r 0.01, p 0.8) (not shown). Similarly, TPA-stimulated lucigenin-elicited chemiluminescence was significantly correlated with eosinophils abundance (r 0.67, p 0.01) (Figure 5A), fibrosis abundance (r 0.43, p 0.05) (Figure 5B), but not with cell abundance (r 0.27, p 0.18) (Figure 5C). Figure 4. Representative picture of polyp eosinophil infiltration and fibrosis. (A, B) High infiltration (score 3, 50% eosinophils). Eosinophils are characterized by a bilobar nucleus and the presence of red granules in the cytoplasm (hematoxilin eosin staining). (A) Original magnification: 100; (B) original magnification: 40. (C) Low infiltration (score 1, 10% eosinophils) and abundant fibrosis. The hemalun eosine safran staining colors the collagen fibers yellow and identifies fibrosis. Original magnification: 100. Characterization of the Lucigenin-elicited Chemiluminescent Signals from Nasal Polyps Polyp fragments stimulated by 1 M PMA induced an immediate lucigenin-elicited chemiluminescent signal. Representative tracings are shown in Figure 1C. SOD 100 U/ml and DPI 100 M inhibited 90% of the lucigenin-enhanced chemiluminescent signal (Figure 6). In contrast, coincubation with denatured SOD (15 min boiling, data not shown), with catalase (2,000 U/ml), or with N w -nitro-l-arginine methyl ester (L-NAME) (100 M) did not affect the morphology or the

5 Pasto, Serrano, Urocoste, et al.: NO and Superoxide Interactions in Nasal Polyps 149 Figure 6. Characterization of the lucigenin-elicited chemiluminescent signals from nasal polyps. SOD superoxide dismutase (100 U/ml), DPI diphenyleneiodonium (100 M), cat catalase (2,000 U/ml), L-NAME N w -nitro-l-arginine methyl ester (100 M). Data are means of triplicate incubations and representative of two separate experiments. Calculation of the Steady-State Concentration of NO Generated by DETA/NO NOC-18 Calculations revealed that 0.75 mm and 1.5 mm of the DETA NONOate generate steady-state NO concentrations of 0.5 M and 2.5 M, respectively. DISCUSSION The major findings of the present study are as follows: (1) Electron spin resonance (ESR) spectroscopy using DMPO as spin trap, ferricytochrome c reduction, and lucigenin-enhanced chemiluminescence can apparently detect the production from a macrophage cell line RAW Three different NO donors dose dependently inhibited these ESR and lucigeninenhanced chemiluminescent signals. (2) The basal and stimulated production in nasal polyps largely varied among patients, and were correlated to eosinophilic abundance. (3) A slow releasing NO donor, DETA NONOate, dose dependently inhibited lucigenin-enhanced chemiluminescence from TPAstimulated polyp fragments. (4) The NO concentrations generated by DETA/NO NOC-18 were calculated, and those in- Figure 5. Correlation between TPA-stimulated lucigenin-elicited chemiluminescence and (A) polyp cell abundance, (B) polyp eosinophils abundance, and (C) polyp fibrosis. amplitude of the chemiluminescent signal. Altogether, these results demonstrate that the lucigenin-enhanced chemiluminescent signal elicited by PMA-stimulated polyp fragment is mainly due to extracellular, a result analogous to that obtained with RAW Effect of a DETA NONOate on the O 2 -elicited Signals from Fragments of Nasal Polyps Various concentrations of DETA NONOate were added 5 min before the addition of 1 m PMA. As shown in Figure 7, DETA/ NO NOC-18 dose dependently inhibited the lucigenin-enhanced chemiluminescent signal with an EC 50 of approximately 1.5 mm. Figure 7. The dose response effect of DETA/NO NOC-18 was studied on the lucigenin-enhanced chemiluminescence generated by TPA-stimulated polyp fragments. Indicated concentrations of DETA NONOate were added 5 min before the addition of 1 M TPA. Data are expressed as percentage of TPA-stimulated polyp fragments obtained in the absence of NO donor.

6 150 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL hibiting lucigenin-enhanced chemiluminescence were found to be within the range of those in the maxillary paranasal sinuses. The first goal of the present study was to validate a technique allowing the evaluation of generation in cells and ex vivo tissues. Indeed, several factors account for the difficulty in assessing the generation of in biological systems. has a very short half-life due to its spontaneous dismutation, which is accelerated by SOD, and also to its rapid reaction with other radical species such as NO (6, 14). In addition, two of the three major techniques used to assess the production of have recently been questioned. The simplest and easiest method for the detection of is the reduction of ferricytochome c. However, Thomson and coworkers recently reported the oxidation of cytochrome c by peroxynitrite (27). These authors pointed out that formation as measured with the cytochrome c reduction technique can be underestimated when NO is simultaneously generated at a comparable rates. This is why we did not determine this technique to study the effect of NO donors on production. Lucigenin luminescence has been widely used to assess the production of. However, the reliability of this technique was also recently questioned (16 18). Far fewer studies have used ESR spectroscopy because this technique requires an expensive tool and is time consuming compared with the two previous ones. In the absence of NO, we found that ESR spectroscopy using DMPO as spin trap, ferricytochrome c reduction, and lucigenin-enhanced chemiluminescence were apparently correlated and quite suitable for assessing the high production from a macrophage cell line RAW In addition, three NO donors (sodium nitroprusside, DETA/NO NOC-18, and SNAP) dose dependently inhibited the amplitude of the ESR and of the lucigenin-enhanced chemiluminescence signals from RAW Thus, lucigenin-enhanced chemiluminescence, which is more sensitive than ESR, was subsequently applied to assess production in fragments of nasal polyps. Large variations of lucigenin-elicited chemiluminescence were found among patients, together with heterogeneous inflammatory cell and eosinophil abundance. Interestingly, both basal and TPAstimulated lucigenin-elicited chemiluminescence were significantly correlated with eosinophils abundance, but not with cell abundance. This correlation is probably the consequence of the high production of eosinophils, this type of phagocytic cells being the most rich in NADPH oxidase (28). NO influences availability by at least two mechanisms. First, because NO and both contains an unpaired electron, both species inactivate each other. Second, NO prevents the activation of NADPH oxidase by inhibiting its assembly process (29). In the present study, the NO donor was added before stimulating the phagocytic cells with TPA to mimic the in vivo conditions (where both mechanisms are acting in concert). Concentrations of DETA/NO NOC-18 applied before TPA stimulation of phagocytic NADPH oxidase dose dependently inhibited the TPA-stimulated lucigenin-elicited chemiluminescence from nasal polyp fragments, with an EC 50 of approximately 1.5 mm. At equilibrium, NO concentrations of ppm were measured in normal maxillary sinuses (8) and are equivalent to a concentration of M in the aqueous phase (30). Concentrations of 0.75 mm and 1.5 mm DETA/NO NOC-18 were calculated to generate steady-state NO concentrations of 0.5 M (10 ppm) and 2.5 M (50 ppm), respectively. Thus, NO concentrations present in maxillary sinuses partially inhibit (approximately 20 40%) the TPA-stimulated lucigenin-elicited chemiluminescence, that is, phagocytic production. The high concentration reached in the paranasal sinus cavities is probably due to the fact that the cavity is almost closed, and thus, the NO concentration can attain values close to the M range. In contrast, in our experiments, the endogenous production of NO by the fragments of nasal polyp rapidly diffused into the air. Under these in vitro conditions, the concentration of NO due to endogenous production did not reach a level high enough to inhibit endogenous superoxide production, as demonstrated by the lack of effect of the NO synthase inhibitor L-NAME (Figure 6). It has been shown that the NO concentrations found in normal sinuses are sufficient to inhibit the growth of several bacteria (9, 31). This reinforces the idea that under normal conditions, NO represents the first line of defense of the paranasal sinuses. Indirect evidence is supported by the dramatic decrease of nasal NO concentration in patients with Kartagener s syndrome (referred to as an immobile cilia syndrome and characterized by situs inversus, sinusitis, and bronchiectasis) (32, 33). However, the sensitivity of the various pathogens to NO, ROS, peroxynitrite, and the synergism and/or antagonism between these two mechanisms of defense require further study (31), and understanding of their respective roles in the pathophysiology of paranasal sinuses probably requires refined modeling. Finally, myeloperoxidase of eosinophils represents an alternative and potentially prominent mechanism of protein nitration (15). This important question about the link between these mechanisms and the pathobiology of the paranasal sinuses also deserves specific treatment. In conclusion, it appears that the paranasal sinuses are an unusual anatomic site where NO accounts for the first line of nonspecific host defense, which could interfere and counteract the second line of defense constituted by the phagocytic cells.the NO concentrations found in paranasal sinuses appear to be within the critical range to inactivate the production of by phagocytes. In vivo, this scheme could be complicated by additional events such as (1) allergic rhinitis increases in nasal NO concentration (33, 34), which can be normalized upon glucocorticoid treatment (35); (2) the potential generation of peroxynitrite, as the respective amounts of NO and reach stoichiometry near 1:1, a condition favorable for the generation of peroxynitrite (14); and (3) the inhibition of apoptosis of certain phagocytic cells such as eosinophils by NO (36). All three mechanisms could contribute to the chronicity of the inflammatory process and thus to the pathophysiology of polyposis. All these events should be analyzed in future studies. References 1. Furchgott RF, Vanhoutte PM. Endothelium-derived relaxing and contracting factors. FASEB J 3: Ignarro LJ. Endothelium-derived nitric oxide: actions and properties. FASEB J 1989;3: Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329: Nathan C. Inducible nitric oxide synthase: what difference does it make? J Clin Invest 1997;100: Chanock S, El Benna J, Smith R, Babior B. The respiratory burst oxidase. J Biol Chem 1994;269: Fridovich I. Superoxide anion radical, superoxide dismutases, and related matters. J Biol Chem 1997;272: Guo F, De Raeve H, Rice T, Stuehr D, Thunnissen F, Erzurum S. Continuous nitric oxide synthesis by inducible nitric oxide synthase in normal airway epithelium in vivo. Proc Natl Acad Sci USA 1995;92: Lundberg J, Farkas-Szallasi T, Weitzberg E, Rinder J, Lidholm J, Änggård A, Hökfelt J, Lundberg JM, Alving K. High nitric oxide production in human paranasal sinuses. Nature Med 1995;1: Mancinelli R, McKay C. Effects of nitric oxide and nitrogen dioxide on bacterial growth. Appl Environ Microbiol 1983;46:

7 Pasto, Serrano, Urocoste, et al.: NO and Superoxide Interactions in Nasal Polyps Jain B, Rubinstein I, Robbins R, Leise K, Sisson J. Modulation of airway epithelial cell ciliary beat frequency by nitric oxide. Biochem Biophys Res Commun 1993;191: Kaliner M, Osguthorpe D, Fireman P, Anon J, Georgitis J, Davis M, Nacero R, Kennedy D. Sinusitis: bench to bedside. J Allergy Clin Immunol 1997;99:S829 S Thébaud B, Arnal J, Mercier J, Dinh-Xuan A. Inhaled and exhaled nitric oxide. Cell Mol Life Sci 1999;55: Rubbo H, Darley-Usmar V, Freeman B. Nitric oxide regulation of tissue free radical injury. Chem Res Toxicol 1996;9: Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrate: the good, the bad, and the ugly. Am J Physiol 1996;271:C1424 C Van der Vliet A, Eiserich J, Shigenaga M, Cross C. Reactive nitrogen species and tyrosine nitration in the respiratory tract. Am J Respir Crit Care Med 1999;160: Liochev SI, Fridovich I. Lucigenin (Bis-N-methylacridinium) as a mediator of superoxide anion production. Arch Biochem Biophys 1997;337: Liochev SI, Fridovich I. Lucigenin as mediator of superoxide production: revisited. Free Rad Biol Med 1998;25: Vasquez-Vivar J, Hogg N, Pritchard KA, JR, Martasek P, Kalyanaraman B. Superoxide anion formation from lucigenin: an electron spin resonance spin-trapping study. FEBS Letters 1997;403: Buettner GR, Oberley LW. Consideration in the spin trapping of superoxide and hydroxyl radicals in aqueous systems using 5,5-dimethyl-1- pyrroline-1-oxide. Biochem Biophys Res Commun 1978;83: Souchard J, Barbacanne M, Margeat E, Maret A, Nepveu F, Arnal J. EPR detection of extracellular superoxide anion released by cultured endothelial cells. Free Rad Res 1998;29: Steinbrecher UP. Role of superoxide in endothelial-cell modification of low-density lipoproteins. Biochim Biophys Acta 1988;959: Kharitonov V, Sundquist A, Sharma V. Kinetics of nitric oxide autoxidation in aqueous solution. J Biol Chem 1994;269: Stieffel E. Differential equations. In: An introduction to numerical mathematics. New York: Academic Press; p Halliwell B, Gutteridge JMC. Detection of free radicals and other reactive species: trapping and fingerprinting. DMPO and PBN. In: Halliwell B, Gutteridge JMC, editors. Free radicals in biology and medicine, 3rd ed. Oxford: Clarendon Press; p Mason RP, Morehouse KM. Spin trapping the ideal method for measuring oxygen radical formation? In: Rice-Evans C, Halliwell B, editors. Free radicals: methodology and concepts. London: Richelieu; p Finkelstein E, Rosen GM, Rauckman EJ, Paxton J. Spin trapping of superoxide. Mol Pharmacol 1979;16: Thomson L, Trujillo M, Telleri R, Radi R. Kinetics of cytochrome c2+ oxidation by peroxynitrite: implications for superoxide measurements in nitric oxide-producing biological systems. Arch Biochem Biophys 1995;319: Someya A, Nishijima K, Nunoi H, Irie S, Nagaoka I. Study of the superoxide-producing enzyme of eosinophils and neutrophils comparison of the NADPH oxidase components. Arch Biochem Biophys 1997; 345: Clancy R, Leszczynska-Piziak J, Abramson S. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide production via a direct action on the NADPH oxidase. J Clin Invest 1992;90: Petit J, Nicaise M, Lepoivre M, Guissani A, Lemaire G. Protection by glutathione against the antiproliferative effects of nitric oxide. Biochem Pharmacol 1996;52: Kaplan S, Lacanster J Jr, Basford R, Simmons R. Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide. Infect Immun 1996;64: Lundberg J, Weitzberg E, Nordvall S, Kuylenstierna R, Lundberg J, Alving K. Primarily nasal origin of exhaled nitric oxide and absence in Kartagener s syndrome. Eur Respir J 1994;7: Arnal J-F, Flores P, Rami J, Murris-Espin M, Bremont F, Pasto M, Aguilla I, Serrano E, Didier A. Nasal nitric oxide concentration in paranasal sinus inflammatory diseases. Eur Respir J 1999;13: Arnal JF, Didier A, Rami J, M Rini C, Charlet JP, Serrano E, Bescombes JP. Nasal nitric oxide is increased in allergic rhinitis. Clin Exp Allergy 1997;27: Kharitonov S, Rajakulasingam K, O Connor B, Durham S, Barnes P. Nasal nitric oxide is increased in patients with asthma and allergic rhinitis and may be modulated by nasal glucocorticoids. J Allergy Clin Immunol 1997;99: Hebestreit H, Dibbert B, Balatti I, Braun D, Shapowal A, Blase K, Simon HV. Disruption of Fas receptor signaling by nitric oxide in eosinophils. J Exp Med 1998;187:

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