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Expression of inducible nitric oxide synthase in antrochoanal polyps
International Journal of Pediatric Otorhinolaryngology (2003) 67, 383 /388
www.elsevier.com/locate/ijporl
Expression of inducible nitric oxide synthase in antrochoanal polyps ¨ zcana,*, Duygu Du ¨ru Cengiz O ¨¸ smez Apab, Yavuz Selim Pataa, Kemal Go ¨ra, Yu ¨cel Akbas¸a a b
Department of Otorhinolaryngology, School of Medicine, Mersin University, Mersin, Turkey Department of Pathology, School of Medicine, Mersin University, Mersin, Turkey
Received 18 August 2002; received in revised form 3 December 2002; accepted 4 December 2002
KEYWORDS Antrochoanal polyp; Inducible NOS; Nitric oxide; Nasal polyp; Immunohistology
Summary Objective: Antrochoanal polyp (ACP) is a polypoid lesion originating from the maxillary sinus, emerging from the ostium and extending to the choana. Although the etiologic causes of ACP are not known completely, it presents a fairly uniform clinical appearance. Nitric oxide (NO) has an important role in non-specific immunoreactions and inflammation in various tissues and has a main regulatory role in airway function and seems to involve in pathomechanism of several respiratory system diseases. NO is synthesized by the effect of three isoforms of nitric oxide synthase i.e. inducible NOS (iNOS). Some studies revealed that ACP has some different characteristics from the ordinary nasal polyps. In the present study, in order to compare ACP with allergic and non-allergic nasal polyps (NANP), we detected and localized the iNOS expression and also evaluate some histological parameters. Methods: Twenty-six cases were chosen from the files and four controls were used. The cases were separated as follows: normal inferior turbinate mucosa as control (n /4) ACPs (n /8), allergic nasal polyps (ANP) (n /9), and NANP (n /9). Fivemicrometer thick sections were prepared from the paraffin sections of polyps and normal nasal mucosa to quantify the iNOS expression. iNOS protein was observed in the cytoplasm of epithelial and stromal inflammatory cells. iNOS protein expression in the groups was assessed according to the intensity of staining and histomorphological parameters, oedema, lymphocytic and eosinophilic cell infiltration were detected semi quantitatively. Results: iNOS expressions, either stromal or epithelial, were not different from each other among the four groups. Although it is not statistically significant, we noted that ANP and ACP frequently showed moderate and severe iNOS protein expression in epithelial and stromal parts when compared with NANPs and controls. Also, iNOS expression was significantly higher in the stroma of the ANPs than NANPs (P /0.012). Conclusions: ACPs and ANPs have frequently showed moderate and significant epithelial and stromal iNOS expression. Further studies are needed in large groups to elucidate differences between ACP and the other nasal polyps. – 2003 Elsevier Science Ireland Ltd. All rights reserved.
*Corresponding author. Address: Fındıkpınarı Caddesi, Bugi Sitesi, No: 28 Kuyuluk, 33200 Mersin, Turkey. Tel.: /90-324-3374300; fax: /90-324-3374305. ¨ zcan). E-mail address: [email protected] (C. O 0165-5876/03/$ - see front matter – 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0165-5876(02)00405-6
¨ zcan et al. C. O
384
1. Introduction
2. Material and methods
Nitric oxide (NO) plays a major role in nonspecific immunoreactions, and inflammation in a variety of tissues [1,2]. NO has a main regulatory role in airway function and seems to involve in pathomechanism of several respiratory system diseases as an important inflammatory mediator and epithelium might be the source of NO production. It was first determined in endothelial cells, and later on described in many cell types like epithelial cells [3 /6]. NO is synthesized from Larginine by the effect of nitric oxide synthase (NOS). The NOS exists in three forms, endothelial NOS, neuronal NOS and inducible NOS (iNOS). iNOS has been detected not only in epithelium but also in macrophages, fibroblasts, neutrophils, endothelium, and vascular smooth muscle [7]. Antrochoanal polyp (ACP) can be defined as a soft tissue lesion originating from the maxillary sinus with a pedicle attached to the inner wall of the maxillary antrum, emerging from the natural or the accessory ostium and extending to the choana through the nasal cavity [8,9]. Nasal polyp is a hypertrophy of the mucous membrane, which covers the ethmoid sinuses and the lateral wall of the nasal fossa [10]. ACPs account for 4 /6% of all nasal polyps in the human population. However, this proportion increases to 33% in the pediatric group [11]. Although the etiologic causes of ACPs are unclear, they present a fairly uniform clinical picture. It has been reported that the ACP might be associated with inflammatory or allergic diseases but the role of allergy in the etiopathogenesis is not clear [12]. Cytokine and arachidonic acid metabolism analysis of nasal polyps have shown that ACP has different features from the usual nasal polyps [13,14]. The paucity of submucous glands in the ACP indicates that the ACP may have little relationship with pathophysiologic events responsible for the development of ordinary nasal polyps [12]. It has been suggested that NO has to be considered in the pathogenesis of nasal polyps [15]. Ramis et al. [3] found that nasal polyps contained higher levels of total NOS activity than normal nasal mucosa. In a recent report, scanning electron microscopic examination of ACPs revealed some differences from the usual nasal polyps [16]. To the best of our knowledge; there has been no reported study about the iNOS expression of ACP in the English language literature until now. In this study, we aimed to detect and evaluate iNOS expression and some histological parameters in ACP and, allergic and non-allergic nasal polyps (NANP).
2.1. Case selection Twenty six cases were chosen from the files and four controls were used. The cases were separated as follows: The controls were normal nasal mucosa taken from the inferior turbinate of the nonsmoker patients who underwent septoplasty operation using cup forceps under general anaesthesia (n /4). Also, ACPs (n /8), allergic nasal polyps (ANP) (n /9), and NANP (n /9) were chosen as study groups.
2.2. Histomorphological evaluation The histomorphological parameters, edema, lymphocytic and eosinophilic infiltration, were detected semi quantitatively as; mild (/), moderate (//), severe (///).
2.3. Immunohistochemical assay Five-micrometer thick sections were prepared from the paraffin sections of polyps and normal nasal mucosa to quantify the iNOS expression. The avidin /biotin complex immunoperoxidase staining system was used. Primary antibodies; rabbit antiiNOS, a synthetic peptide derived from the extreme C-terminus of the human iNOS protein (Zymed Laboratories, Lexington, KY); with a dilution of 1:1000 were used. The sections were deparaffinized in xylene through ethanol to phosphate-buffered saline (pH 7.2). Hydrogen peroxide (3%) was applied to block endogenous peroxidase activity for 30 min. The slides incubated in citrate buffer were heated in microwave owen for 5 min. After waiting 20 min, they were removed and ultra V block (Labvision, Fremont, CA) was added. Primary antibody for iNOS was applied and incubated overnight in a moist chamber at 4 8C. The slides were subsequently incubated in biotinylated goat anti-polyvalent (Labvision) for 10 min and in streptavidin peroxidase (Labvision) for 20 min. Finally, AEC substrate system (Labvision) was applied for about 3 min. After each incubation, the sections were rinsed with distilled and tap water. The tissue was counterstained with Mayer’s hematoxyline stain. All slides were covered with a cover slip after mounting in buffered glycerine.
2.4. iNOS morphometric determination iNOS protein was observed in the cytoplasm of epithelial and stromal inflammatory cells. iNOS protein expression in the groups was assessed
Expression of inducible NOS in antrochoanal polyps
according to the intensity of staining. The best stained areas were found in each case, and the percentage of the iNOS stained cells were recorded and graded as follows; B/2% absent, score 0; 2 / 10% weak, score 1; 10 /50%, intermediate, score 2; /50% strong, score 3.
385
Table 2 groups
Stromal cell iNOS expression scores in
Groups
0 ACP(n /8) ANP (n /9) NANP (n /9) Control (n /4)
2.5. Statistical analysis
Scores
3 3 8 3
1 (37.5)a (33.3) (88.9) (75.0)
3 1 1 1
2 (37.5) (11.1) (11.1) (25.0)
3
2 (25.0) 0 2 (22.2) 3 (33.3) 0 0 0 0
Mann /Whitney U and Kruskal /Wallis tests were used in the statistical analysis of histological and immunohistologic findings and P B/0.05 values are accepted as statistically significant.
3. Results iNOS expressions, both stromal and epithelial, were not different from each other among the four groups (Inferior turbinate mucosa as control, ACP, ANP and NANP). (Kruskal /Wallis test, P /0.517). Although it is not statistically significant, we noted that ACP and ANP groups frequently showed moderate and severe iNOS protein expression in epithelial and stromal parts when compared with NANP and inferior turbinate mucosa (Tables 1 and 2) (Figs. 1 and 2a and b). Also, iNOS expression was significantly higher in the stroma of the ANP than NANP (Mann /Whitney U test, P /0.012) (Fig. 3a and b). There was a statistically significant difference among the four groups for the lymphocytic and eosinophilic infiltration (Kruskal /Wallis test, P / 0.001). Lymphocytic infiltration was higher in ACP and NANP comparing with the ‘control’ group (Mann /Whitney U test, P /0.004 and 0.011, respectively). Lymphocytic infiltration was also higher in ACP group comparing with ANP group (Mann /Whitney U test, P /0.009) (Fig. 4a). Eosinophilic infiltration was higher in ACPs, NANPs, and ANPs comparing with normal nasal mucosa (control group) (Mann /Whitney U test, P /0.011, 0.006, and 0.001, respectively). EosinoTable 1 Surface epithelial iNOS expression scores in groups Groups
Scores 0
ACP (n /8) ANP (n /9) NANP (n /9) Control (n /4) a
3 5 6 3
1 (37.5)a (55.6) (66.7) (75.0)
3 1 2 1
(37.5) (11.1) (22.2) (25)
Case number (% within group).
2
3
2 (25) 2 (25) 1 (11.1) 0
0 1 (11.1) 0 0
Fig. 1 Strong iNOS expression was seen in stromal cells in ACP ( /200, iNOS).
philic infiltration was higher in NANP and ANP groups comparing with ACPs (Mann /Whitney U test, P /0.006 and 0.0001) (Fig. 4b). Eosinophilic infiltration was not different in ANP and NANP groups.
4. Discussion The present study revealed that ACP and ANP frequently showed moderate and severe iNOS protein expression in epithelial and stromal parts although it is not statistically significant. Both lymphocytic and eosinophilic infiltration was higher in all of the polyps groups comparing with control nasal mucosa. The lymphocytic infiltration was higher and eosinophilic infiltration was lower in the ACPs comparing with the ANPs. Recent studies suggested that NO, an endogenous vasodilator and inflammatory mediator, may be generated in the nasal cavity. NO is synthesized by a group of NOS and three isoforms have been identified in the inflammatory conditions in human [17]. iNOS may be expressed in a variety of cells in inflammation, and regulated in response to stimuli, such as proinflammatory cytokines [1,18 /20]. In the specimens of patients with chronic rhinitis, moderate immunostaining for iNOS was seen in the
386
Fig. 2 iNOS expression in glandular epithelium ( /200, iNOS) (a), and in surface epithelium (large arrow) (b) ( / 400, iNOS) in ACP.
surface epithelium, glandular, inflammatory and vascular endothelial cells [19]. Nasal polyps generally originate from the ethmoid cells and consist of an epithelial lining surrounding underlying edema, glandular hyperplasia, fibrosis and eosinophilic infiltration is the characteristic histological feature. The polypoid stroma is highly edematous with a varying density of inflammatory cells. The pathophysiology of nasal polyps remains poorly understood [21]. They have long been associated with rhinitis, aspirin sensitivity, cystic fibrosis, Kartagener’s syndrome and asthma [10,21]. However, the role of allergy in the etiology and pathogenesis of nasal polyps is controversial [12,21,22]. ACP is a large, firm, fleshy, myxoid polyp, having a long stalk. Histologically, it is an inflammatory polyp covered with ciliated cylindrical epithelium [12,22]. The stroma is usually edematous and highly vascular, composed of loose connective tissue mainly infiltrated with plasma cells and a few eosinophils. Most authors noted that its histopathology did not differ significantly from the usual nasal polyps. The etiologic causes of ACP have not been fully determined. However, chronic sinusitis and allergy have been accused [12,14,16,22].
¨ zcan et al. C. O
Fig. 3 Strong stromal iNOS expression in ANP ( /200, iNOS) (a) ( /200, iNOS) and weak iNOS expression in NANP (b) ( /400, iNOS).
The increased nasal NO may originate from two sources: the epithelial or inflammatory cells. Recent studies suggested that eosinophils express iNOS and release NO. Nasal NO production may be associated with eosinophil recruitment but does not correlate directly with eosinophil number [6]. In patients with chronic rhinitis, there has been strong correlation between the extent of inflammation and the expression of iNOS [19]. It has been stated that iNOS expression is upregulated in nasal polyps, especially in epithelial layer. It was reported that inflammatory mechanisms associated with the formation of nasal polyps induce the expression of iNOS in nasal mucosal line [17]. NO synthesis is enhanced locally at sites of inflammation but the role of NO in inflammation is not clear. Mechanisms that are involved in the induction of iNOS expression in nasal cavity are also unknown. However, it was assumed that locally produced cytokines may play an important role in induction of epithelial cells by iNOS [20]. Inflammatory mediators derived from infiltrating inflammatory cells, or nasal epithelial cells have been implicated in the pathogenesis of nasal polyps [2].
Expression of inducible NOS in antrochoanal polyps
387
decongestants have an inhibitory effect on iNOS activity [4]. Generally, local and systemic steroids, and nasal decongestant agents are given to patients with nasal polyps, preoperatively and routine therapy. This may be the reason why some of our cases were not shown significant epithelial and stromal iNOS expression. In conclusion, this study shows that ACP and ANP have frequently showed moderate or significant epithelial and stromal iNOS expression although there were not any statistically significant differences. Further studies are needed to elucidate the possible mechanisms of NO in pathogenesis of ACP and differences between ACP and nasal polyps in larger groups.
Acknowledgements We thank Arzu Kanık, Ph.D., and Cengiz Pata, M.D., for the statistical analysis of data and for the technical assistance.
References Fig. 4 Heavy lymphocytic infiltration (thin arrow) in ACP (a), ( /400, H&E), and eosinophils (large arrow) in ANP (b), ( /400, H&E).
It has been known that there was a relationship between NO and allergic diseases [23 /25]. Kharitonov et al. [23] showed that nasal NO was significantly elevated in untreated allergic rhinitis patients compared with normal persons or individuals treated with topical steroids. It has been also reported that iNOS expression of the epithelial cells in allergic rhinitis patients’ mucosa had increased and it was assumed that stimulated secretion of cytokines during allergic reactions was responsible for the increased iNOS expression [24]. We observed that four of nine ANP cases have iNOS expression in the epithelium but six of nine cases have iNOS expression in the stroma. Watkins et al. [17] revealed that nasal turbinate did not demonstrate immunohistological staining for iNOS but opposite findings were also reported [19]. Our finding was parallel to Watkins et al. [17] findings for the iNOS expression of inferior turbinate mucosa. Recently, it was reported that polyps of aspirin-sensitive patients had a greater amount of iNOS and suggested that NO has a role in the aetiopathogenesis of nasal polyps with aspirin-sensitive patients [26]. iNOS is inhibited by glucocorticoids and topical steroids and the level of nasal derived NO is reduced [23]. It was also shown that nasal
[1] S. Moncada, R.M. Palmer, R.M.J. Ferriege, A.E. Higgs, Nitric oxide: physiology, pathophysiology and pharmacology, Pharmacol. Rev. 43 (1991) 109 /142. [2] A. Hess, W. Bloch, J. Rockher, K. Addicks, E. Stennert, L. Michel, In vitro expression of inducible nitric oxide synthase in the nasal mucosa of guinea pigs after incubation with lipopolysaccharides or cytokines, Eur. Arch. Otorhinolaryngol. 255 (1998) 448 /453. [3] I. Ramis, J. Lorente, J. Roselio-Catafau, P. Quesada, E. Gelpi, O. Bulbena, Differential activity of nitric oxide synthase in human nasal mucosa and nasal polyp, Eur. Respir. J. 9 (1996) 202 /206. [4] G.J. Westeweld, H.P. Voss, R.M. van der Hee, G.J.N. de Haan-Koelewijn, G.J.M. den Harog, R.A. Scheeren, A. Bast, Inhibition of nitric oxide synthase by nasal decongestants, Eur. Respir. J. 16 (2000) 437 /444. [5] R.M.J. Palmer, A.G. Ferrige, S. Moncada, Nitric oxide accounts for the biological activity of endothelium-derived relaxing factor, Nature 327 (1987) 524 /526. [6] T. Hanazawa, J.D. Antuni, S.A. Kharitonow, P.J. Barnes, Intranasal administration of eotaxin increases nasal eosinophils and nitric oxide in patients with allergic rhinitis, J. Allergy Clin. Immunol. (1999) 58 /64. [7] P.G. Djupesland, J.M. Chatkin, W. Qian, J.S.J. Haight, Nitric oxide in the nasal airway: a new dimension in otolaryngology, Am. J. Otolaryngol. 22 (2001) 19 /32. [8] J.G. Batsakis, N. Sneige, Choanal angiomatous polyps of the sinonasal tract, Ann. Otol. Rhinol. Laryngol. 101 (1992) 623 /625. ¨ zcan, M. U ¨ nal, K. Go [9] C. .O ¨ru ¨r, Y.S. Pata, Antrokoanal polipli 14 olgunun incelenmesi, Kulak Burun Bogaz Ihtis Derg 9 (2002) 188 /192. [10] M. Chmielik, A. Wasiutnyski, J. Maldyk, B. Wachulski, D. Bartoszek, Histological analysis of nasal polyps in children, Int. J. Pediatr. Otorhinolaryngol. 60 (2001) 131 /133.
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388 [11] S. Bas¸ak, C.Z. Karaman, A. Akdilli, K.K. Metin, Surgical approaches to antrochoanal polyps in children, Int. J. Pediatr. Otorhinolaryngol. 46 (1998) 197 /205. [12] Y.-G. Min, J.W. Chung, J.-S. Shin, J.G. Chi, Histologic structure of antrochoanal polyps, Acta Otolaryngol. 115 (1995) 543 /547. [13] H. Saito, N. Honda, T. Yamada, S. Mori, S. Fujieda, T. Saito, Intractable pediatric chronic sinusitis with antrochoanal polyp, Int. J. Pediatr. Otorhinolaryngol. 54 (2000) 111 /116. [14] Y.J. Jang, C.K. Rhee, C.K. Oh, H.G. Ryoo, H.G. Kim, M. Ha, Arachidonic acid metabolites in antrochoanal polyp and nasal polyp associated with chronic paranasal sinusitis, Acta Otolaryngol. 120 (2000) 531 /534. [15] A. Heb, W. Bloch, J. Rocker, S. Peters, E. Stennert, K. Addicks, O. Michel, Distribution of nitric oxide-synthases under physiological and pathophysiological conditions, HNO 48 (2000) 489 /495. [16] J. Skladzien, J.A. Litwin, M. Nowogrodzka-Zagorska, W. Wierzchowski, Morphological and clinical characteristics of antrochoanal polyps: comparison with chronic inflammation-associated polyps of the maxillary sinus, Auris Nasus Larynx 28 (2001) 137 /141. [17] D.N. Watkins, R.H. Lewis, K.A. Basclain, P.H. Fisher, D.J. Peroni, M.J. Garlepp, P.J. Thompson, Expression and localization of the inducible isoform of nitric oxide synthase in nasal polyp epithelium, Clin. Exp. Allergy 28 (1998) 211 /219. [18] P.J. Conway, N.S. Jones, The nose and nitric oxide: a review, Clin. Otolaryngol. 25 (2000) 337 /341.
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[19] K. Furukawa, D.G. Harrison, D. Saleh, H. Shennib, F.P. Chagnon, A. Giaid, Expression of nitric oxide synthase in nasal mucosa, Am. J. Crit. Care Med. 153 (1996) 847 /850. [20] W. Li, J. Lin, G.L. Adams, S.K. Juhn, Expression of inducible nitric oxide synthase (iNOS) in middle ear epithelial cells by IL-1 beta and TNF-alpha, Int. J. Pediatr. Otorhinolaryngol. 55 (2000) 91 /98. [21] N. Mygind, R. Dahl, C. Bachert, Nasal polyposis, eosinophil dominated inflammation, and allergy, Thorax 55 (Suppl. 2) (2000) 79 /83. [22] J.M. Chen, M. Schloss, M.E. Azouz, Antro-choanal polyp: a 10-year retrospective study in the pediatric population with a review of the literature, J. Otolaryngol. 18 (1989) 168 /172. [23] S.A. Kharitonov, K. Rajakulalasingam, B. O’Connor, S.R. Durham, P.J. Barnes, Nasal nitric-oxide increased in patients with asthma and allergic rhinitis and may be modulated by nasal glucocorticoids, J. Allergy Clin. Immunol. 99 (1997) 58 /64. [24] H. Kawamoto, S. Takeno, K. Yajin, Increased expression of inducible nitric oxide synthase in nasal epithelial cells in patients with allergic rhinitis, Laryngoscope 109 (1999) 2015 /2020. [25] J.F. Arnal, A. Didier, J. Rami, C. M’rini, J.P. Charlet, E. Serrano, J.P. Besombes, Nasal nitric oxide is increased in allergic rhinitis, Clin. Exp. Allergy 27 (1997) 358 /362. [26] A. Parikh, G.K. Scadding, P. Gray, M.G. Belvisi, J.A. Mitchell, High levels of nitric oxide synthase activity are associated with nasal polyp tissue from aspirin-sensitive asthmatics, Acta Otolaryngol. 122 (2002) 302 /305.
www.elsevier.com/locate/ijporl
Expression of inducible nitric oxide synthase in antrochoanal polyps ¨ zcana,*, Duygu Du ¨ru Cengiz O ¨¸ smez Apab, Yavuz Selim Pataa, Kemal Go ¨ra, Yu ¨cel Akbas¸a a b
Department of Otorhinolaryngology, School of Medicine, Mersin University, Mersin, Turkey Department of Pathology, School of Medicine, Mersin University, Mersin, Turkey
Received 18 August 2002; received in revised form 3 December 2002; accepted 4 December 2002
KEYWORDS Antrochoanal polyp; Inducible NOS; Nitric oxide; Nasal polyp; Immunohistology
Summary Objective: Antrochoanal polyp (ACP) is a polypoid lesion originating from the maxillary sinus, emerging from the ostium and extending to the choana. Although the etiologic causes of ACP are not known completely, it presents a fairly uniform clinical appearance. Nitric oxide (NO) has an important role in non-specific immunoreactions and inflammation in various tissues and has a main regulatory role in airway function and seems to involve in pathomechanism of several respiratory system diseases. NO is synthesized by the effect of three isoforms of nitric oxide synthase i.e. inducible NOS (iNOS). Some studies revealed that ACP has some different characteristics from the ordinary nasal polyps. In the present study, in order to compare ACP with allergic and non-allergic nasal polyps (NANP), we detected and localized the iNOS expression and also evaluate some histological parameters. Methods: Twenty-six cases were chosen from the files and four controls were used. The cases were separated as follows: normal inferior turbinate mucosa as control (n /4) ACPs (n /8), allergic nasal polyps (ANP) (n /9), and NANP (n /9). Fivemicrometer thick sections were prepared from the paraffin sections of polyps and normal nasal mucosa to quantify the iNOS expression. iNOS protein was observed in the cytoplasm of epithelial and stromal inflammatory cells. iNOS protein expression in the groups was assessed according to the intensity of staining and histomorphological parameters, oedema, lymphocytic and eosinophilic cell infiltration were detected semi quantitatively. Results: iNOS expressions, either stromal or epithelial, were not different from each other among the four groups. Although it is not statistically significant, we noted that ANP and ACP frequently showed moderate and severe iNOS protein expression in epithelial and stromal parts when compared with NANPs and controls. Also, iNOS expression was significantly higher in the stroma of the ANPs than NANPs (P /0.012). Conclusions: ACPs and ANPs have frequently showed moderate and significant epithelial and stromal iNOS expression. Further studies are needed in large groups to elucidate differences between ACP and the other nasal polyps. – 2003 Elsevier Science Ireland Ltd. All rights reserved.
*Corresponding author. Address: Fındıkpınarı Caddesi, Bugi Sitesi, No: 28 Kuyuluk, 33200 Mersin, Turkey. Tel.: /90-324-3374300; fax: /90-324-3374305. ¨ zcan). E-mail address: [email protected] (C. O 0165-5876/03/$ - see front matter – 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0165-5876(02)00405-6
¨ zcan et al. C. O
384
1. Introduction
2. Material and methods
Nitric oxide (NO) plays a major role in nonspecific immunoreactions, and inflammation in a variety of tissues [1,2]. NO has a main regulatory role in airway function and seems to involve in pathomechanism of several respiratory system diseases as an important inflammatory mediator and epithelium might be the source of NO production. It was first determined in endothelial cells, and later on described in many cell types like epithelial cells [3 /6]. NO is synthesized from Larginine by the effect of nitric oxide synthase (NOS). The NOS exists in three forms, endothelial NOS, neuronal NOS and inducible NOS (iNOS). iNOS has been detected not only in epithelium but also in macrophages, fibroblasts, neutrophils, endothelium, and vascular smooth muscle [7]. Antrochoanal polyp (ACP) can be defined as a soft tissue lesion originating from the maxillary sinus with a pedicle attached to the inner wall of the maxillary antrum, emerging from the natural or the accessory ostium and extending to the choana through the nasal cavity [8,9]. Nasal polyp is a hypertrophy of the mucous membrane, which covers the ethmoid sinuses and the lateral wall of the nasal fossa [10]. ACPs account for 4 /6% of all nasal polyps in the human population. However, this proportion increases to 33% in the pediatric group [11]. Although the etiologic causes of ACPs are unclear, they present a fairly uniform clinical picture. It has been reported that the ACP might be associated with inflammatory or allergic diseases but the role of allergy in the etiopathogenesis is not clear [12]. Cytokine and arachidonic acid metabolism analysis of nasal polyps have shown that ACP has different features from the usual nasal polyps [13,14]. The paucity of submucous glands in the ACP indicates that the ACP may have little relationship with pathophysiologic events responsible for the development of ordinary nasal polyps [12]. It has been suggested that NO has to be considered in the pathogenesis of nasal polyps [15]. Ramis et al. [3] found that nasal polyps contained higher levels of total NOS activity than normal nasal mucosa. In a recent report, scanning electron microscopic examination of ACPs revealed some differences from the usual nasal polyps [16]. To the best of our knowledge; there has been no reported study about the iNOS expression of ACP in the English language literature until now. In this study, we aimed to detect and evaluate iNOS expression and some histological parameters in ACP and, allergic and non-allergic nasal polyps (NANP).
2.1. Case selection Twenty six cases were chosen from the files and four controls were used. The cases were separated as follows: The controls were normal nasal mucosa taken from the inferior turbinate of the nonsmoker patients who underwent septoplasty operation using cup forceps under general anaesthesia (n /4). Also, ACPs (n /8), allergic nasal polyps (ANP) (n /9), and NANP (n /9) were chosen as study groups.
2.2. Histomorphological evaluation The histomorphological parameters, edema, lymphocytic and eosinophilic infiltration, were detected semi quantitatively as; mild (/), moderate (//), severe (///).
2.3. Immunohistochemical assay Five-micrometer thick sections were prepared from the paraffin sections of polyps and normal nasal mucosa to quantify the iNOS expression. The avidin /biotin complex immunoperoxidase staining system was used. Primary antibodies; rabbit antiiNOS, a synthetic peptide derived from the extreme C-terminus of the human iNOS protein (Zymed Laboratories, Lexington, KY); with a dilution of 1:1000 were used. The sections were deparaffinized in xylene through ethanol to phosphate-buffered saline (pH 7.2). Hydrogen peroxide (3%) was applied to block endogenous peroxidase activity for 30 min. The slides incubated in citrate buffer were heated in microwave owen for 5 min. After waiting 20 min, they were removed and ultra V block (Labvision, Fremont, CA) was added. Primary antibody for iNOS was applied and incubated overnight in a moist chamber at 4 8C. The slides were subsequently incubated in biotinylated goat anti-polyvalent (Labvision) for 10 min and in streptavidin peroxidase (Labvision) for 20 min. Finally, AEC substrate system (Labvision) was applied for about 3 min. After each incubation, the sections were rinsed with distilled and tap water. The tissue was counterstained with Mayer’s hematoxyline stain. All slides were covered with a cover slip after mounting in buffered glycerine.
2.4. iNOS morphometric determination iNOS protein was observed in the cytoplasm of epithelial and stromal inflammatory cells. iNOS protein expression in the groups was assessed
Expression of inducible NOS in antrochoanal polyps
according to the intensity of staining. The best stained areas were found in each case, and the percentage of the iNOS stained cells were recorded and graded as follows; B/2% absent, score 0; 2 / 10% weak, score 1; 10 /50%, intermediate, score 2; /50% strong, score 3.
385
Table 2 groups
Stromal cell iNOS expression scores in
Groups
0 ACP(n /8) ANP (n /9) NANP (n /9) Control (n /4)
2.5. Statistical analysis
Scores
3 3 8 3
1 (37.5)a (33.3) (88.9) (75.0)
3 1 1 1
2 (37.5) (11.1) (11.1) (25.0)
3
2 (25.0) 0 2 (22.2) 3 (33.3) 0 0 0 0
Mann /Whitney U and Kruskal /Wallis tests were used in the statistical analysis of histological and immunohistologic findings and P B/0.05 values are accepted as statistically significant.
3. Results iNOS expressions, both stromal and epithelial, were not different from each other among the four groups (Inferior turbinate mucosa as control, ACP, ANP and NANP). (Kruskal /Wallis test, P /0.517). Although it is not statistically significant, we noted that ACP and ANP groups frequently showed moderate and severe iNOS protein expression in epithelial and stromal parts when compared with NANP and inferior turbinate mucosa (Tables 1 and 2) (Figs. 1 and 2a and b). Also, iNOS expression was significantly higher in the stroma of the ANP than NANP (Mann /Whitney U test, P /0.012) (Fig. 3a and b). There was a statistically significant difference among the four groups for the lymphocytic and eosinophilic infiltration (Kruskal /Wallis test, P / 0.001). Lymphocytic infiltration was higher in ACP and NANP comparing with the ‘control’ group (Mann /Whitney U test, P /0.004 and 0.011, respectively). Lymphocytic infiltration was also higher in ACP group comparing with ANP group (Mann /Whitney U test, P /0.009) (Fig. 4a). Eosinophilic infiltration was higher in ACPs, NANPs, and ANPs comparing with normal nasal mucosa (control group) (Mann /Whitney U test, P /0.011, 0.006, and 0.001, respectively). EosinoTable 1 Surface epithelial iNOS expression scores in groups Groups
Scores 0
ACP (n /8) ANP (n /9) NANP (n /9) Control (n /4) a
3 5 6 3
1 (37.5)a (55.6) (66.7) (75.0)
3 1 2 1
(37.5) (11.1) (22.2) (25)
Case number (% within group).
2
3
2 (25) 2 (25) 1 (11.1) 0
0 1 (11.1) 0 0
Fig. 1 Strong iNOS expression was seen in stromal cells in ACP ( /200, iNOS).
philic infiltration was higher in NANP and ANP groups comparing with ACPs (Mann /Whitney U test, P /0.006 and 0.0001) (Fig. 4b). Eosinophilic infiltration was not different in ANP and NANP groups.
4. Discussion The present study revealed that ACP and ANP frequently showed moderate and severe iNOS protein expression in epithelial and stromal parts although it is not statistically significant. Both lymphocytic and eosinophilic infiltration was higher in all of the polyps groups comparing with control nasal mucosa. The lymphocytic infiltration was higher and eosinophilic infiltration was lower in the ACPs comparing with the ANPs. Recent studies suggested that NO, an endogenous vasodilator and inflammatory mediator, may be generated in the nasal cavity. NO is synthesized by a group of NOS and three isoforms have been identified in the inflammatory conditions in human [17]. iNOS may be expressed in a variety of cells in inflammation, and regulated in response to stimuli, such as proinflammatory cytokines [1,18 /20]. In the specimens of patients with chronic rhinitis, moderate immunostaining for iNOS was seen in the
386
Fig. 2 iNOS expression in glandular epithelium ( /200, iNOS) (a), and in surface epithelium (large arrow) (b) ( / 400, iNOS) in ACP.
surface epithelium, glandular, inflammatory and vascular endothelial cells [19]. Nasal polyps generally originate from the ethmoid cells and consist of an epithelial lining surrounding underlying edema, glandular hyperplasia, fibrosis and eosinophilic infiltration is the characteristic histological feature. The polypoid stroma is highly edematous with a varying density of inflammatory cells. The pathophysiology of nasal polyps remains poorly understood [21]. They have long been associated with rhinitis, aspirin sensitivity, cystic fibrosis, Kartagener’s syndrome and asthma [10,21]. However, the role of allergy in the etiology and pathogenesis of nasal polyps is controversial [12,21,22]. ACP is a large, firm, fleshy, myxoid polyp, having a long stalk. Histologically, it is an inflammatory polyp covered with ciliated cylindrical epithelium [12,22]. The stroma is usually edematous and highly vascular, composed of loose connective tissue mainly infiltrated with plasma cells and a few eosinophils. Most authors noted that its histopathology did not differ significantly from the usual nasal polyps. The etiologic causes of ACP have not been fully determined. However, chronic sinusitis and allergy have been accused [12,14,16,22].
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Fig. 3 Strong stromal iNOS expression in ANP ( /200, iNOS) (a) ( /200, iNOS) and weak iNOS expression in NANP (b) ( /400, iNOS).
The increased nasal NO may originate from two sources: the epithelial or inflammatory cells. Recent studies suggested that eosinophils express iNOS and release NO. Nasal NO production may be associated with eosinophil recruitment but does not correlate directly with eosinophil number [6]. In patients with chronic rhinitis, there has been strong correlation between the extent of inflammation and the expression of iNOS [19]. It has been stated that iNOS expression is upregulated in nasal polyps, especially in epithelial layer. It was reported that inflammatory mechanisms associated with the formation of nasal polyps induce the expression of iNOS in nasal mucosal line [17]. NO synthesis is enhanced locally at sites of inflammation but the role of NO in inflammation is not clear. Mechanisms that are involved in the induction of iNOS expression in nasal cavity are also unknown. However, it was assumed that locally produced cytokines may play an important role in induction of epithelial cells by iNOS [20]. Inflammatory mediators derived from infiltrating inflammatory cells, or nasal epithelial cells have been implicated in the pathogenesis of nasal polyps [2].
Expression of inducible NOS in antrochoanal polyps
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decongestants have an inhibitory effect on iNOS activity [4]. Generally, local and systemic steroids, and nasal decongestant agents are given to patients with nasal polyps, preoperatively and routine therapy. This may be the reason why some of our cases were not shown significant epithelial and stromal iNOS expression. In conclusion, this study shows that ACP and ANP have frequently showed moderate or significant epithelial and stromal iNOS expression although there were not any statistically significant differences. Further studies are needed to elucidate the possible mechanisms of NO in pathogenesis of ACP and differences between ACP and nasal polyps in larger groups.
Acknowledgements We thank Arzu Kanık, Ph.D., and Cengiz Pata, M.D., for the statistical analysis of data and for the technical assistance.
References Fig. 4 Heavy lymphocytic infiltration (thin arrow) in ACP (a), ( /400, H&E), and eosinophils (large arrow) in ANP (b), ( /400, H&E).
It has been known that there was a relationship between NO and allergic diseases [23 /25]. Kharitonov et al. [23] showed that nasal NO was significantly elevated in untreated allergic rhinitis patients compared with normal persons or individuals treated with topical steroids. It has been also reported that iNOS expression of the epithelial cells in allergic rhinitis patients’ mucosa had increased and it was assumed that stimulated secretion of cytokines during allergic reactions was responsible for the increased iNOS expression [24]. We observed that four of nine ANP cases have iNOS expression in the epithelium but six of nine cases have iNOS expression in the stroma. Watkins et al. [17] revealed that nasal turbinate did not demonstrate immunohistological staining for iNOS but opposite findings were also reported [19]. Our finding was parallel to Watkins et al. [17] findings for the iNOS expression of inferior turbinate mucosa. Recently, it was reported that polyps of aspirin-sensitive patients had a greater amount of iNOS and suggested that NO has a role in the aetiopathogenesis of nasal polyps with aspirin-sensitive patients [26]. iNOS is inhibited by glucocorticoids and topical steroids and the level of nasal derived NO is reduced [23]. It was also shown that nasal
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