Protein nitration in chronic sinusitis and nasal polyposis: Role of eosinophils

Protein nitration in chronic sinusitis and nasal polyposis: Role of eosinophils JULIO FREIRE BERNARDES, MD, JICHUAN SHAN, MD, MARC TEWFIK, DAVID H. EIDELMAN, MD, Montreal, Quebec, Canada

BSC,

OBJECTIVES: To investigate the possible role of eosinophil peroxidase (EPO) in 3-nitrotyrosine (3NT) formation. STUDY DESIGN AND SETTING: Observational study employing immunocytochemistry to assess the presence of 3NT, inducible nitric oxide synthase (iNOS), eosinophils, mast cells, neutrophils, and lymphocytes in ethmoid sinus mucosal biopsies from normal controls and subjects with allergic and nonallergic chronic sinusitis and nasal polyposis. RESULTS: 3NT was more evident in biopsies from sinusitis patients (2.67 ⴞ 0.14, n ⴝ 21) than in healthy mucosa (0.43 ⴞ 0.2, n ⴝ 7, P < 0.01), but scores in atopic and nonatopic subjects were similar. Colocalization studies confirmed that 3NT was largely confined to eosinophils. No relationship was found between 3NT and other immune cells. 3NT detection was not correlated with the amount of immunostaining for iNOS. SIGNIFICANCE: Chronic sinusitis is accompanied by 3NT formation, which is largely restricted to the eosinophils, and likely driven by the action of eosinophil peroxidase, rather than by nitric oxide levels. EBM rating: B-2. (Otolaryngol Head Neck Surg 2004;131:696-703.)

inflammatory thickening and polypoid changes of the sinus mucosa with marked eosinophilia.1 When particularly severe,2 the condition may be complicated by the development of nasal polyps, which pathologically contain cystically dilated, inspissated mucous glands and dedifferentiated epithelium. Regardless of the atopic status of individuals, eosinophils dominate the inflammatory cell influx3 in most cases of sinusitis that are not related to bacterial infection.1 Chronic sinusitis is also associated with altered nitric oxide (NO) metabolism.4-7 NO is a ubiquitous biologically active radical produced in large quantities by healthy human paranasal sinus epithelium. Indeed, NO concentrations in healthy sinuses can rise close to the highest permissible environmental pollution levels,8 leading to the hypothesis that9 NO contributes to sinus host defense by virtue of its antimicrobial properties10 and ability to upregulate ciliary activity.4,8 The most important source of NO in the respiratory tract is the inducible isoform of nitric oxide synthase (NOS), termed iNOS or NOS type II. In the upper and lower airways, iNOS is primarily expressed in epithelial cells and macrophages, and is upregulated by cytokines, microbes, or microbial products,8 permitting NO production to increase markedly response to infection and in inflammatory states. In the presence of high concentrations of superoxide anion (O2⫺), NO can be converted to peroxynitrite (ONOO⫺) or other reactive intermediates,4 that may induce covalent modifications in proteins and other biomolecules with the potential to alter cellular function and promote tissue injury. One such modification yields 3-nitrotyrosine (3NT), and detection of this adduct in proteins is now commonly used as a marker of NO-mediated oxidative stress.8,10 Formation of 3NT has been detected in severe perennial nasal allergy in association with NO production5 and similar observations have been made in asthma,11,12 suggesting the possibility that tyrosine nitration plays a pathogenetic role in eosinophilic airway diseases. It is usually inferred that detection of protein nitration in these diseases reflects the action of ONOO⫺ that is formed from increased production of NO in the presence of abundant O2⫺. Another pathway for the production of nitrotyrosine may also be of importance, however. Peroxidases, particularly eosinophil peroxidase (EPO), are capable of promoting 3NT formation though mechanisms that are not driven by the

C hronic sinusitis is characterized by signs and symptoms of sinus inflammation that persist for at least 4 weeks after initiating appropriate medical therapy in the absence of an intervening acute episode.1 The pathological hallmark of most forms of chronic sinusitis is From the Meakins-Christie Laboratories, Respiratory Division, Department of Medicine, McGill University Health Centre (Drs Bernardes, Shan, Tewfik, Hamid, and Eidelman) and the Department of Otolaryngology, SMDBJewish General Hospital (Drs Bernardes, Twefik, and Frenkiel), Montreal, Canada. Presented in abstract form at the American Thoracic Society International Conference, Atlanta, GA, May 20, 2002. Supported by Canadian Institutes for Health Research and the JT Costello Foundation. Reprint requests: David Eidelman, MD, Meakins-Christie Laboratories, 3626 St. Urbain Street, Montreal QC, Canada H2X 2P2; e-mail, david.eidelman@ mcgill.ca. 0194-5998/$30.00 Copyright © 2004 by the American Academy of Otolaryngology–Head and Neck Surgery Foundation, Inc. doi:10.1016/j.otohns.2004.05.013

696

QUTAYBA HAMID,

MD, PHD,

SAUL FRENKIEL,

MD,

and

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Table 1. Subject characteristics Groups

n

Mean age

Range

Smokers

Gender

Control Nonatopic Atopic Total

7 6 15 28

51.1 39.7 47.5 46.7

40-62 26-66 27-66 26-66

1 0 8 9

4M/3F 4M/2F 8M/7F 16M/12F

Allergy skin testing

Aspirin sensitivity

Asthma

Negative Negative Positive

0 0 3 3

1 1 5 7

The patient numbers (n), mean age and range, male (M)/female (F) ratio and number of subjects with the other characteristics are shown.

rate of NO production but which depend on the enzymatic activity of the peroxidase.13,14 Given the prominence of eosinophils in chronic sinusitis, it is possible that detection of 3NT in this disorder reflects the activity of eosinophils rather than simply augmented production of NO. To understand the role of the eosinophil in protein nitration in sinusitis, we used immunohistochemistry to study biopsies from subjects with chronic sinusitis and control subjects. Our findings strongly suggest that the detection of 3NT in this disorder is closely related to the presence of eosinophils themselves, presumably through the action of EPO, rather than to excessive NO production. METHODS Patients Biopsies of ethmoid sinus mucosa were obtained from 28 patients (16 males, 12 females), with a mean age of 47 (range, 26 – 66 years of age). Seven patients undergoing surgery and without any history of nasal inflammation served as a control group. The control group was compared with 21 patients suffering from nasal polyposis and chronic sinusitis. All patients who attended the Sir Mortimer B. Davis Jewish General Hospital Otolaryngology clinic were evaluated for possible inclusion. Occasional random eligible patients were approached for participation in the study. Patients using oral or topical steroids were excluded. Nasal biopsies were harvested during the course of therapeutically indicated surgery. All procedures employed in this study were reviewed and approved by the research ethics committee of the Sir Mortimer B. Davis Jewish General Hospital. The diagnosis of chronic sinusitis was based on clinical symptoms, paranasal CT scan, and nasal endoscopic inspection. Atopy was defined based on skin testing. Subjects positive for at least 1 of 12 common aeroallergens (ragweed, plantain, grasses, rye, trees, birch, Alternaria, hormodendrum, mites, dust, cats, and dogs) on skin-prick testing were defined as atopic. The patients were classified into 3 groups: (1) nonatopic controls, (2) atopic patients with chronic sinusitis, or (3) nonatopic patients with chronic sinusitis. One patient in the control group had asthma and another was

a smoker. Among the nonatopic subjects, 1 patient had asthma. Among the atopic subjects, 3 patients had a triad of aspirin sensitivity, asthma, and nasal polyposis; 5 patients had asthma; and 8 were smokers (Table 1). The diagnoses of aspirin sensitivity and asthma were based on the medical history. Tissue Harvesting Nasal mucosa and nasal polyps were removed surgically and stored in ice-cold Eagle’s Minimum Essential Medium with Spinner Modification (S-MEM; GIBCO, Rockville, MD) supplemented with penicillin and streptomycin for less than 2 hours before being processed. The tissue was then cut with a razor blade into fragments, blocked in liquid nitrogen, and conserved at ⫺80°C. Cryostat sections were made, fixed in ethanol/methanol solution (60:40), and submitted to immunocytochemistry, or stored again at ⫺80°C for later use. Nitrotyrosine and iNOS Cryostat sections (8-␮m thick) were thawed, washed twice in Tris-buffered saline (TBS; 0.05 M pH 7.6) and placed inside a humidified chamber. We verified the loss of immunostaining following treatment of specimens with dithionite solution (500 mM sodium hydrosulfite dissolved in 100 mM sodium borate; Sigma, Oakville, Ontario) for 30 minutes. All slides were incubated with universal blocking solution (Dako, Mississauga, Ontario, Canada), for 10 minutes at room temperature, followed by the primary 3NT antibody (Upstate Biotechnology, Lake Placid, NY) or iNOS antibody (Rb antimouse iNOS pAB, Transduction Laboratories, Mississauga, Ontario, Canada) diluted in antibody diluting buffer (Dako, 1:400 and 1:200 respectively), overnight at 4°C. After washing in TBS 3 times, slides were incubated with secondary antibody (biotinylated Swine X Rabbit IgG, Dako, in a 1:200 dilution) for 50 minutes at room temperature. Control specimens developed without the primary antibody were used to verify that nonspecific binding by the secondary antibody was not detectable. After washing again with TBS, streptavidin alkaline phosphatase (Dako) was added at a 1:200 dilution, and incubated for 50 minutes.

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The slides were washed with TBS and developed using Fast Red TR (Sigma, Oakville, Ontario, Canada) dissolved in substrate for alkaline phosphate and filtered through a 0.2 filter, and was visually monitored for color change (approximately 10-15 min). Washing in TBS and then tap water terminated the reaction. The slides were then counterstained with hematoxylin, placed in lithium carbonate for 20 seconds, and rinsed in tap water. Crystal mount (Biomeda Corporation, Foster City, CA) was then applied. The slides were dried at 37°C overnight, and mounted with a coverslip. Inflammatory Cells For detection of inflammatory cells, cryostat sections were treated initially as above and then incubated overnight at 4°C with mouse anti-human major basic protein antibody (Biodesign International, Saco, ME, 1:30 dilution) for eosinophils, mouse anti-human elastase antibody (Dako, 1:100 dilution) for neutrophils, mouse anti-human tryptase antibody (Chemicon International, Temecula, CA, 1:250 dilution) for mast cells and anti-human CD4 antibody (Dako, 1:100 dilution). After washing in TBS 3 times, slides were treated with the secondary antibody (rabbit antimMouse IgG, Dako; 1:60 dilution) for 30 minutes at room temperature. After again washing with TBS, mouse APAAP (Dako) at a 1:200 dilution was added and incubated for 30 minutes. The slides were washed and developed with Fast Red as above. Washing in TBS and then tap water terminated the reaction. Slides were then counterstained in hematoxylin, placed in lithium carbonate for 20 seconds, and mounted as described earlier. Immunofluorescence Studies For confocal immunofluorescent studies, cryostat sections were thawed and washed 3 times in PBS for 15 minutes. Slides were then placed inside a humidified chamber and blocked with universal blocking solution for 30 minutes. Slides were then incubated overnight at 4°C with primary antibodies as follows: anti-3NT (rabbit anti-human 3NT antibody, Upstate Biotechnology, diluted 1:200), anti-MBP (mouse anti-human major basic protein antibody, diluted 1:30), or anti-EPO (mouse anti-eosinophil peroxidase, Research Diagnostics, Flanders, NJ, diluted 1:50). We ensured specificity of binding by performing control experiments in which the first antibody was excluded. On the next day, the slides were washed again 3 times in PBS for 15 minutes, and then incubated with FITC-conjugated secondary antibody for anti-3NT (swine anti-rabbit IgG antibody, Dako, diluted 1:50), or incubated with a second conjugated antibody for anti-MBP and anti-EPO (Alexa Fluor 568 goat anti-mouse IgG, Molecular Probes, Eugene,

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OR, diluted 1:100) at room temperature for 60 minutes. Slides were then washed 1 final time with PBS, and mounted with glass coverslips using Perma Fluor Aqueous Mounting Medium (Immunon, Pittsburgh, PA). Double Immunofluorescence To determine the contribution of eosinophils and eosinophil peroxidase to 3NT formation, we performed double staining. Slides were pretreated as described above and incubated overnight at 4°C with mixture of the primary antibodies (anti-3NT and anti-MBP or anti3NT and anti-EPO), maintaining the appropriate final dilution of each antibody as shown above. The slides were washed 3 times in PBS for 15 minutes and then incubated with swine anti-rabbit FITC-conjugated secondary antibody for 60 minutes at room temperature to detect 3NT. After again washing with PBS, goat antimouse Alexa 568 IgG was added and incubated for more 60 minutes at room temperature to detect either MBP or EPO. No cross-reactivity between the secondary antibodies was detected in control experiments. Slides were then processed as above. Scoring To compare groups of biopsies we adopted a semiquantitative scoring procedure that measured the intensity of immunostaining as follows. A score of 0 was assigned for negative staining, 1 for minimal staining, 2 for a moderate staining, and 3 for an intense staining. This approach is similar to that used by other investigators.6 All scoring was done in a blinded fashion and 2 trained observers reviewed all slides. Concordance between the 2 observers was excellent (Pearson r2 ⫽ 0.98). Statistical Analysis All data are expressed as mean ⫾ SEM. Immunostaining scores were compared with the unpaired Student t-test. Differences were considered significant when P ⬍ 0.05. Statistical comparisons were carried out using the Sigma plot 4.0 for Windows (SPSS Science, Chicago, IL). RESULTS Cellular Infiltration Nasal polyps were characterized by evidence of moderate to severe inflammatory changes in the submucosa associated with edema, hypertrophy of submucosal glands and squamous metaplasia of the surface epithelium, consistent with chronic inflammation (Table 2). On conventional histological examination, the inflammatory infiltrate appeared to be dominated by eosinophils, which was confirmed by immunohistochemistry (data not shown). As expected, chronic sinusitis and polyposis were associated

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Fig 1. Nitrotyrosine and MBP, group data. Staining for both nitrotyrosine (closed bars) and the eosinophil marker MBP (open bars) was increased in the mucosa of both allergic and non-allergic subjects compared to the control group (P ⬍ 0.01). There was no statistically significant difference between allergic and nonallergic subjects. Bars are means ⫹ SEM.

with moderate to intense MBP staining. The immunostaining intensity score for allergic and non-allergic groups (2.80 ⫾ 0.11 and 2.67 ⫾ 0.21, respectively) were significantly higher than in the mucosa of normal controls (0.29 ⫾ 0.18, P ⬍ 0.01). This contrasted with findings for other cells. Only weak to moderate elastase staining was seen in all 3 groups (1.29 ⫾ 0.36, 0.83 ⫾ 0.17, and 1.60 ⫾ 0.31, respectively). The intensity of staining for mast cells, detected as tryptase positivity, did not to differ among groups, with moderate to strong staining in controls, nonallergic, and allergic patients (2.00 ⫾ 0.21,2.17 ⫾ 0.17, and 2.07 ⫾ 0.15, respectively). However, the increase in MBP staining was mirrored by evidence of increased intensity of CD4 staining in the allergic group (2.0 ⫾ 0.27) compared to the normal control (0.33 ⫾ 0.33, P ⫽ 0.02). No statistically significant increase was found in the nonallergic (1.80 ⫾ 0.73) group, however. Nitrotyrosine Immunostaining There was abundant staining for 3NT in both groups of subjects with chronic sinusitis (allergic, 2.60 ⫾ 0.19; nonallergic, 2.83 ⫾ 0.17), with scores that were significantly greater than those seen in the mucosa of control subjects (0.43 ⫾ 0.20). 3NT was detected on the surface epithelium, in submucosal glands and particularly around inflammatory cells (see Fig 5A). There was no statistically significant difference between allergic and nonallergic subjects. When subjects were grouped by pathology, specimens from subjects with nasal polyposis showed significantly more intense staining for 3NT than did specimens from normal controls (2.67 ⫾ 0.14 vs. 0.43 ⫾ 0.20, P ⬍ 0.0001).

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Fig 2. Relationship between nitrotyrosine and MBP staining by subject. Nitrotyrosine score is displayed on the abscissa and MBP staining on the ordinate. Each symbol represents 1 patient. There was a consistent tendency for MBP and nitrotyrosine staining to be correlated on a subject-by-subject basis for all 3 groups (control, atopic, and nonatopic).

Nitrotyrosine and Eosinophils There was a close relationship between 3NT staining and eosinophils in all groups (see Fig 2). When double immunofluorescence was performed we observed colocalization (see Fig 5, yellow color) between 3NT (FITC green) and either MBP (Alexa 568 red) or EPO (Alexa 568 red). There was little evidence of green fluorescence outside of cells, suggesting that most 3NT staining in polyposis is related to the eosinophil. No association between 3NT staining and other the other cell types studied was noted. Nitric Oxide Synthase iNOS was mainly localized to surface epithelium, submucosal glands and associated inflammatory cells in all 3 groups. However, normal mucosa presented with weak to moderate staining (0.80 ⫾ 0.20), whereas moderate to strong staining was found in nonatopic and atopic subjects (1.40 ⫾ 0.51 and 2.50 ⫾ 0.27, respectively). iNOS staining was significantly higher in chronic sinusitis patients than among controls. DISCUSSION The goal of this study was to investigate the possible role of the eosinophil in the development of protein nitration in chronic sinusitis. As expected, in biopsies from both atopic and nonatopic individuals

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Fig 3. Immunocytochemistry score for inflammatory cells. Data is shown for control subjects (upper panel), nonallergic subjects (middle panel), and allergic subjects (lower panel). MBP staining was significantly higher (P ⬍ 0.01) in both allergic (n ⫽ 15) and nonallergic groups (n ⫽ 6), respectively, than among controls (n ⫽ 7). Although weak to moderate staining for neutrophils and mast cells was found in all 3 groups, there were no statistically significant differences among groups. Staining for CD4-positive lymphocytes was increased in allergic subjects (P ⬍ 0.05), but not nonallergic subjects, compared with controls.

with chronic sinusitis we observed evidence of both eosinophilic inflammation and 3NT formation. It has been suggested that the detection of 3NT in sinusitis5 and asthma11,12 is a consequence of excessive nitric oxide production in a milieu where O2⫺ is also produced in high concentration. In the present study, however, we found not only that the detection of 3NT and the presence of eosinophils related to each

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other for the groups overall, but colocalization studies demonstrated that most of the 3NT is restricted to the eosinophils themselves (Fig 5). Our findings thus underscore the importance of eosinophils in the pathogenesis of nitric oxide mediated protein transformation and support a role for eosinophil peroxidase in 3NT formation. Chronic sinusitis is a syndrome of unknown etiology often associated with asthma and allergic disease. In its severest form, chronic sinusitis is marked by the development of nasal polyposis, an inflammatory disorder of the mucous membranes in the nose and paranasal sinuses presenting as pedunculated smooth, semitranslucent and round masses of inflamed mucosa prolapsing into the nose.1 As with chronic sinusitis generally, eosinophils are the dominant inflammatory cell in nasal polyposis except in cystic fibrosis, where neutrophils predominate.1 In the present study, we found immunocytochemical evidence of eosinophilic infiltration, primarily in the submucosa, in both atopic and nonatopic individuals (Fig 1). Eosinophils are thought to potentially contribute to the pathogenesis of chronic sinusitis through their capacity to release a variety of toxic proteins like MBP and eosinophil cationic protein (ECP), highly cationic proteins that potentially contribute to tissue injury.15 Eosinophils also are an important source of oxidants, largely through the action of NADPH oxidase and eosinophil peroxidase.14,16 Moreover, although epithelial cells are thought to be the major source of NO production in the sinuses, human eosinophils may also express iNOS11 and potentially contribute to NO production directly. We observed that in both atopic and nonatopic subjects the increase in 3NT generally mirrored the increase in detection of MBP, a marker for eosinophils. Given that inflammatory conditions like asthma11,12 and allergic rhinitis,7 are often associated with evidence of 3NT formation,17 it is not surprising that biopsies from our subjects with sinusitis showed evidence of 3NT formation (Fig 2). Whereas previous studies did not attempt to identify the cells responsible for protein nitration, we found a close association between detection of 3NT and detection of MBP immunoreactivity at the level of individual subjects. Irrespective of atopic status, those with the highest degree of MBP immunoreactivity also tended to have the most 3NT immunoreactivity (Fig 3). This was confirmed using double immunofluorescence confocal microscopy (Fig 5), which demonstrated that much of the 3NT immunoreactivity was confined to the eosinophils themselves, intimately associated with immunoreactivity for both MBP and EPO (Fig 5). Our findings raise questions about the significance of detecting 3NT in sinusitis, as well as related condi-

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Fig 4. Immunocytochemistry score for inducible nitric oxide synthase (iNOS). There was evidence of weak to moderate positivity for iNOS in control subjects (n ⫽ 7). Although nonallergic subjects (n⫽7) showed evidence of increased staining for iNOS, there was a significant increase in iNOS immunoreactivity in allergic subjects (n ⫽ 6, P ⬍ 0.01).

tions such as asthma.11 The colocalization of 3NT with eosinophils (Fig 5) casts doubt on the importance of peroxynitrite formation as the mechanism underlying protein nitration in these disorders. Instead, these data point to EPO, which has the capacity to promote 3NT formation,13,16,18 through a reaction that depends on NO2⫺ and which is independent of peroxynitrite.8,14 Our findings suggest that previous reports of 3NT in the context of sinusitis5 may reflect the presence of activated eosinophils rather than excessive NO and O2⫺ production per se. In addition, these results suggest that the detection of 3NT in tissue,11,12 sputum,19 or exhaled breath20 may only reflect the presence of peroxidase laden granulocytes, rather than the underlying rate of production of peroxynitrite. To the extent that peroxynitrite formation is driven by increased availability of NO, we would expect some relationship between 3NT and iNOS immunoreactivity, at least outside of eosinophils. Although iNOS immunoreactivity was increased in patients with atopic sinusitis (Fig 4), there was no difference between nonatopic subjects and controls while 3NT was increased in both sinusitis groups to a similar degree. Furthermore, iNOS staining was largely confined to the mucosa, where little 3NT was detected. Indeed, we were surprised by the relative lack of evidence of iNOS upregulation in subjects with sinusitis. This may have related to metaplasia and sloughing of the nasal epithelium that we frequently observed, possibly resulting in less iNOS staining than might otherwise be expected. Of interest, nasal exhaled NO has at times been reported to be paradoxically decreased in chronic sinusitis21and in

Fig 5. Immunofluorescence staining: representative examples of confocal images of cryostat sections. Examples of nitrotyrosine immunofluorescence in (A) nonatopic nasal polyposis (⫻100, FITC-conjugated anti-3NT), (B) MBP immunofluorescence in nonatopic nasal polyposis (⫻100, Alexa 568-conjugated anti-MBP), and (C) immunofluorescence for EPO (⫻400, Alexa 568-conjugated anti-EPO). There was extensive evidence of eosinophilia and nitrotyrosine formation in all cases of sinusitis, both atopic and nonatopic. Examples of colocalization with double immunofluorescence staining, showing the close relationship between nitrotyrosine and (D) MBP (⫻100, FITC-conjugated anti-MBP; Alexa 568-conjugated anti3NT) or (E) eosinophil peroxidase (⫻400, FITC-conjugated anti-EPO and Alexa 568-conjugated anti-3NT). Yellow color demonstrates colocalization. The remaining panels demonstrate examples of iNOS immunofluorescence in control (⫻100, F), atopic (⫻100, G), and nonatopic (⫻100, H) subjects. iNOS was mainly localized to surface epithelium, submucosal glands, and around inflammatory cells in all 3 groups, which was strongest in atopic subjects.

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Table 2. Intensity of immunostaining in nasal mucosa as group means

Control Nonatopic Atopic

Anti-3NT

Anti-MBP

Antielastase

Antitryptase

Anti-CD4

0.43 ⫾ 0.20 2.83 ⫾ 0.17* 2.60 ⫾ 0.19*

0.29 ⫾ 0.18 2.67 ⫾ 0.21* 2.80 ⫾ 0.11*

1.29 ⫾ 0.36 0.83 ⫾ 0.17 1.60 ⫾ 0.31

2.00 ⫾ 0.22 2.17 ⫾ 0.17 2.07 ⫾ 0.15

0.33 ⫾ 0.33 1.80 ⫾ 0.73 2.00 ⫾ 0.27*

Results reflect means ⫾ SEM for immunodetection of nitrotyrosine (anti-3NT), eosinophils (anti- MBP), neutrophils (anti-elastase), mast cells (anti-tryptase), and T-helper lymphocytes (anti-CD4). *P ⬍ 0.05 compared with the control group.

children with acute sinusitis.22 This has been interpreted as a result of impaired flow of NO from the sinuses due to ostial obstruction. It is also possible that, given the presence of nitrotyrosine staining, some of the NO is consumed in the formation of oxidative derivatives. Although our findings tend to exclude a role for peroxynitrite in the 3NT formation in chronic sinusitis, peroxynitrite is still be important. Peroxynitrite is rapidly formed from O2⫺ and molecular NO at a very high reaction rate. Because of repeated cycling of congestion and decongestion of the nasal mucosa that leads to fluctuations in mucosal blood flow (5), O2⫺ production in chronic sinusitis may be very prominent. Polyps typically originate in the upper part of the nose, lateral to the middle turbinate, and around the openings of the ethmoid and maxillary sinuses, where mucosal surfaces come into close contact1 and where congestion may easily lead to vascular compromise. The resulting ischemia and subsequent reperfusion promotes O2⫺ generation from endothelial xanthine oxidase in addition to the expected production by NADPH oxidase in inflammatory cells.23 Increased production of peroxynitrite would therefore be expected in chronic sinusitis, potentially altering cellular function for example by influencing intracellular signaling through its capacity to nitrate tyrosine kinases or oxidize phosphatases.24 Such biologically important effects may fall below the threshold of detection by immunohistochemistry and semi-quantitative scoring. Our observations must be interpreted in the light of the limitations inherent in an observational study such as this. No experimental manipulation of the subjects was attempted. Furthermore, detection of 3NT was accomplished solely by immunohistochemistry as there was insufficient material to use other methods. Nevertheless, this approach was sufficient for our primary goal of identifying the source of 3NT and confirming the importance of eosinophils in this phenomenon. Moreover, using double immunofluorescence we confirmed that 3NT is closely related to the MBP and EPO in and immediately surrounding eosinophils. These findings underscore the importance of eosinophils in

mediating oxidative protein transformation and strongly suggest that the detection of 3NT formation in chronic sinusitis reflects the activity of EPO. Future studies aimed at understanding the role of NO mediated tissue injury in sinusitis need to take into account the important contribution of peroxidases to protein nitration. The authors would like to thank Dr. Meri Tulic for her advice and assistance, Ms. Elsa Schotman and Ms. Michelle Robinson for their expert technical help and Ms. Lisa Shearer for her secretarial assistance. REFERENCES 1. Kaliner MA, Osguthorpe JD, Fireman P et al. Sinusitis: bench to bedside. Current findings, future directions. Otolaryngol Head Neck Surg 1997;116(6 Pt 2):S1-20. 2. Lamblin C, Gosset P, Salez F, et al. Eosinophilic airway inflammation in nasal polyposis. J Allergy Clin Immunol 1999;104:85-92. 3. Christodoulopoulos P, Cameron L, Durham S, et al. Molecular pathology of allergic disease. II: Upper airway disease. J Allergy Clin Immunol 2000;105(2 Pt 1):211-23. 4. Pasto M, Serrano E, Urocoste E, et al. Nasal polyp-derived superoxide anion: dose-dependent inhibition by nitric oxide and pathophysiological implications. Am J Respir Crit Care Med 2001;163:145-51. 5. Sato M, Fukuyama N, Sakai M, et al. Increased nitric oxide in nasal lavage fluid and nitrotyrosine formation in nasal mucosa– indices for severe perennial nasal allergy. Clin Exp Allergy 1998;28:597-605. 6. Furukawa K, Harrison DG, Saleh D, et al. Expression of nitric oxide synthase in the human nasal mucosa. Am J Respir Crit Care Med 1996;153:847-50. 7. Kang BH, Chen SS, Jou LS, et al. Immunolocalization of inducible nitric oxide synthase and 3-nitrotyrosine in the nasal mucosa of patients with rhinitis. Eur Arch Otorhinolaryngol 2000;257:242-6. 8. van der Vliet A, Eiserich JP, Shigenaga MK, et al. Reactive nitrogen species and tyrosine nitration in the respiratory tract. Epiphenomena Or a pathobiologic mechanism of disease? Am J Respir Crit Care Med 1999;160:1-9. 9. Lundberg JO, Farkas-Szallasi T, Weitzberg E, et al. High nitric oxide production in human paranasal sinuses. Nat Med 1995; 1:370-3. 10. Silkoff PE, Robbins RA, Gaston B, et al. Endogenous nitric oxide in allergic airway disease. J Allergy Clin Immunol 2000;105:438-48. 11. Saleh D, Ernst P, Lim S, et al. Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid. FASEB J 1998;12:929-37. 12. Kaminsky DA, Mitchell J, Carroll N, et al. Nitrotyrosine formation in the airways and lung parenchyma of patients with asthma. J Allergy Clin Immunol 1999;104(4 Pt 1):747-754.

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13. Iijima H, Duguet A, Eum SY et al. Nitric oxide and protein nitration are eosinophil dependent in allergen-challenged mice. Am J Respir Crit Care Med 2001;163:1233-40. 14. MacPherson JC, Comhair SA, Erzurum SC, et al. Eosinophils are a major source of nitric oxide-derived oxidants in severe asthma: characterization of pathways available to eosinophils for generating reactive nitrogen species. J Immunol 2001;166:5763-72. 15. Hoover GE, Newman LJ, Platts-Mills TA, et al. Chronic sinusitis: risk factors for extensive disease. J Allergy Clin Immunol 1997;100:185-91. 16. Wu W, Chen Y, Hazen SL. Eosinophil peroxidase nitrates protein tyrosyl residues. Implications for oxidative damage by nitrating intermediates in eosinophilic inflammatory disorders. J Biol Chem 1999;274:25933-44. 17. Hanazawa T, Antuni JD, Kharitonov SA, et al. Intranasal administration of eotaxin increases nasal eosinophils and nitric oxide in patients with allergic rhinitis. J Allergy Clin Immunol 2000; 105(1 Pt 1):58-64. 18. Duguet A, Iijima H, Eum SY, et al. Eosinophil peroxidase

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mediates protein nitration in allergic airway inflammation in mice. Am J Respir Crit Care Med 2001;164:1119-26. van der Vliet A, Nguyen MN, Shigenaga MK, et al. Myeloperoxidase and protein oxidation in cystic fibrosis. Am J Physiol Lung Cell Mol Physiol 2000;279:L537-46. Hanazawa T, Kharitonov SA, Barnes PJ. Increased nitrotyrosine in exhaled breath condensate of patients with asthma. Am J Respir Crit Care Med 2000;162(4 Pt 1):1273-6. Lindberg S, Cervin A, Runer T. Nitric oxide (NO) production in the upper airways is decreased in chronic sinusitis. Acta Otolaryngol 1997;117:113-7. Baraldi E, Azzolin NM, Biban P, et al. Effect of antibiotic therapy on nasal nitric oxide concentration in children with acute sinusitis. Am J Respir Crit Care Med 1997;155:1680-3. Li C, Jackson RM. Reactive species mechanisms of cellular hypoxia-reoxygenation injury. Am J Physiol Cell Physiol 2002;282:C227-41. Minetti M, Mallozzi C, Di Stasi A. Peroxynitrite activates kinases of the src family and upregulates tyrosine phosphorylation signaling(1,2). Free Radic Biol Med 2002;33:744.