B-ENT, 2013, 9, 207-216

Inducible nitric oxide synthase (iNOS) in sinonasal polyp pathogenesis
N. Bayar Muluk1, O. K. Arıkan2, P. Atasoy3, R. Kılıç1 and E. Tuna Yalçinozan4
ENT Department, 3Pathology Department, Kırıkkale University, Faculty of Medicine, Kırıkkale, Turkey; 2ENT
Department, Adana Numune Research and Training Hospital, Adana, Turkey; 4ENT Department, Artvin State Hospital,
Artvin, Turkey

1

Key-words. Sinonasal polyp, pathogenesis, inducible nitric oxide synthase (iNOS), glandular, endothelial, iNOS positivity index
Abstract. Inducible nitric oxide synthase (iNOS) in sinonasal polyp pathogenesis. Objectives: We investigated the roleof inducible nitric oxide synthase (iNOS) in the pathogenesis of sinonasal polyps.
Methods: Adult patients (21 men, 3 women) with nasal polyposis underwent functional endoscopic sinus surgery. Nine
adults without polyps (6 men) who underwent septoplasty and/or rhinoplasty served as controls. Polyp specimens camefrom three regions: the maxillary sinus (10), ethmoid sinus (14), and nasal cavity (10). Control group samples (9) camefrom the inferior turbinate. Specimens were evaluated in eight mucosal layers for count and distribution of inflammatory
cells and iNOS expression. An iNOS positivity index (PI) was determined for the epithelium (E), subepithelial layer ofthe lamina propria (SE), and deep paraglandular layer of the mucosa (D).
Results: Polymorphonuclear cell (PMNC) % values of the ethmoid and maxillary sinus and overall ethmoid sinus PI weresignificantly higher in the polyp group. Patients with longer polyp duration, D-perivascular (D-pv), and a higher­Brinkmann index had decreased ethmoid sinus D PIs. However, in older patients and patients with longer polyp duration,
perivascular PIs increased in maxillary sinus SE and D, respectively. Furthermore, as PMNC % and iNOS-PMNC PI
increased, SE_glandular and epithelial_apical iNOS values decreased. In the ethmoid and maxillary sinuses, iNOS_D_
endothelial values increased but decreased in the nasal cavity.
Conclusions: iNOS may play a role in sinonasal polyp pathogenesis, especially in mucosal SE and D layers. Increased
vascular permeability, stromal edema, inflammatory cell migration into the stroma of the mucosa, and increased mucosal
gland secretion may result in polyp formation.

Introduction
Inflammation of the upper airways is among themost common and frequent diseases and a categorythat includes nasal polyposis, associated in manycases with immune response disorders. An inflammatory infiltration of mononuclear, eosinophil,plasma, and mast cells can be found in the histological structure of the polypous as well as tonsillar
mucosa.1
Several mechanisms have been proposed for theformation of nasal polyps, including allergy, mucosal allergy, autonomic imbalance, nitric oxide(NO), superantigens, infection, abnormal trans­
epithelial ion transport, mucopolysaccharide abnormality, mechanical obstruction, and epithelial­rupture. Eosinophils comprise more than 60% of
the cell population. Activated T cells, mast cells,

and plasma cells are also increased compared with
the normal nasal mucosa. The stroma has numerous
mediators, including cytokines, growth factors,­adhesion molecules, and immunoglobulins. BothTh1 and Th2 types of cytokines are upregulated­independent of the atopic status. Increased production of GM-CSF, IL5, RANTES, and eotaxin cancontribute to chronic eosinophilic inflammation by
regulating the migration, survival, and activation ofeosinophils.2,3
NO is a ubiquitous biologically active radical
produced in large quantities by healthy human paranasal sinus epithelium. NO (formula .N = O) is a
simple, inorganic, gaseous free radical with predominant functions as a messenger and effector
molecule. In mammals, NO is synthesized from Larginine by a family of enzymes referred to as thenitric oxide synthases (NOS)4 and is an importantFinancial support: This study was supported by Kirikkale University Scientific Research Projects Unit Funds.

208
cellular signaling molecule with a vital role in many
biological processes.5 NO synthesis is catalyzed by
one of three isoforms of NOS: neuronal NOS, endothelial NOS (eNOS), and inducible NOS (iNOS).6
Moderate immunostaining for iNOS has been
identified in surface epithelium; glandular, inflammatory, and vascular endothelial cells; and smoothmuscle cells in specimens from patients withchronic­ rhinitis only.4-7 In situ hybridization shows
eNOS mRNA expression in sites similar to those
identified by immunohistochemistry whereas themRNA for iNOS predominantly localizes to inflammatory cells.7
In the present study, we investigated the role ofiNOS in the pathogenesis of nasal polyps. The­polyp specimens were evaluated in eight layersof the mucosa for the count and distribution of
inflammatory­ cells and their iNOS expression.
Confounding factors affecting the iNOS positivity
index (PI) levels at the epithelial, subepithelial, anddeep ­layers were also investigated.
Materials and methods
The study was performed in the Otolaryngology
Department of Kirikkale University Faculty of
Medicine. The immunohistochemical staining andlight microscopy examinations were performed by
the university’s pathology department. All stepsof the study were planned and conducted withapproval­ from the Kirikkale University Facultyof Medicine Local Ethics Committee and according
to the principles outlined in the Declaration of
Helsinki­.8
Subjects
The nasal polyp group was selected from patients
examined in the Otolaryngology Department of
Kirikkale University Faculty of Medicine. They
had used topical corticosteroid nasal spray for atleast 6 weeks and until the day of surgery; if thepathology persisted, surgery was performed. This
group consisted of 24 adults (21 men) with nasal
polyps who underwent functional endoscopic sinus
surgery (FESS). Patient ages were between 23≤
and ≤70; the mean age was 46.5 ± 11.2. All patientsin the study group answered a questionnaire andunderwent an ENT examination, endoscopic­examination with 0º and 30º endoscopes, Waters’view radiography, and axial and coronal computed

N. Bayar Muluk et al.
tomography (CT) of the paranasal sinuses.
The control group consisted of nine adults without
nasal polyps (6 men) who underwent a septoplasty.
Their ages ranged between 18≤ and ≤55; the mean
age was 28.22 ± 12.24. They agreed to enter the
study and gave written approval, and all of the­examination processes described above were also
performed for the participants in the control group.
In both groups, there were no other acute or chronic
diseases, such as asthma, chronic obstructive­pulmonary diseases, or acute or chronic sinusitis.
Methods
Questionnaire. The questionnaire covered anterior
and posterior nasal discharge, nasal congestion,
cough, facial and dental pain, halitosis, paroxysmal
nocturnal coughing spells, sore throat, fever, olfactory loss, headache, and ear pain.9 Polyp duration
was assessed by hospital data and patient history,
including the time of first diagnosis of a nasal polypand follow-up data.
Endoscopic examination. Endoscopic examination with 0º and 30º endoscopes was performed in
the Endoscopy Unit of the ENT Department of
Kirikkale University Faculty of Medicine. Discharge(none, clear and thin, thick, purulent); mucosal­status (normoplasia, light hyperplasia with no erythema, hyperplasia),10 anatomic anomalies (septal
deviation, lateral rotation of the uncinate process,turbinate hypertrophy, and other anatomic anomalies);9 and localization and size of the polyps were
examined.
In preoperative nasal endoscopic examination of
the polyp group, appearance of nasal polyps wasstaged according to Lawson (1991).11 Stage 0, no
polyp presented; Stage 1, polyp under medial turbinate seen by endoscopy; Stage 2, protruding polypin medial turbinate seen without endoscopy; andStage 3: massive polyposis.
CT. Localization and size of polyps in the nasal
cavity and paranasal sinuses were evaluated by­axial and coronal sections of the paranasal sinusesin the polyp group. Also investigated were pan-­polyposis, septal deviation, concha bullosa, lateralrotation of the uncinate process, prominent ethmoid
bulla, and other anatomic abnormalities.9
Surgery. All patients with polyps underwent
FESS for nasal polyposis. Biopsies were performed
under general anesthesia with samples obtained
from macroscopically observed polypoid areas.

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iNOS in sinonasal polyp pathogenesis
Specimens including polyp tissue were excisedfrom three regions: the nasal cavity and the maxillary and ethmoid (anterior and posterior) sinuses.
The specimens were examined at x400 magnification under light microscopy, and only slides withhistopathological evidence of polypoid tissue were
included in the study. Tissues that were edematousand rich in vessels, with inflammatory cells andpolypoid development, were included in the study
as the polyp group. Slides suggesting a chronic inflammatory process without polypoid tissue were
excluded. Finally, the study group covered three
regions: ethmoid sinuses (14 specimens), maxillarysinus (10 specimens), and nasal cavity (10 specimens). In the control group, specimens were collected via punch biopsies from inferior turbinates
during septoplasty for a total of nine specimens in
the control group.
Immunohistochemical staining. In the study andcontrol groups, a surgical specimen was examinedwith an immunohistochemical staining techniqueusing a monoclonal antibody against iNOS, nitricoxide synthase, inducible Ab-1 rabbit polyclonal
antibody (Thermo Scientific/LabVision Corporation, Fremont, CA, USA). In each of the surgical
specimens, iNOS positivity was evaluated in 3–4
high-magnification fields under light microscopy
and the mean number of cells in the epithelium (E),subepithelial layer of the lamina propria (SE), anddeep paraglandular layer of the mucosa (D) determined.
Immunohistochemical staining technique. Sections in 5 µm thickness were obtained, transferredto adhesive slides, and dried in an autoclave at 37°C
overnight and at 60°C for 20 minutes. They weredeparaffinized and dehydrated by immersion in xylene twice for 10 minutes and in alcohol twice for10 minutes. The specimen was then incubated in
3% H2O2 for 5 minutes to inhibit endogenous peroxidases. The preparations were transferred into a
citrate-based antigen retrieval solution (Dako; Denmark; pH 6) for iNOS antigens (Thermo Scientific/
LabVision Corporation Neomarkers, Fremont, CA,
USA). The nitric oxide synthase, inducible Ab-1
rabbit polyclonal antibody (Thermo Scientific/LabVision Corporation, Fremont, CA, USA). was used.
All slides were microwaved (750 W) for 5 minutes
two times. Following the instructions in the Shandon Sequenza manual for standardization, the classical streptavidin–avidin/biotin–peroxidase methodand diaminobenzidine chromogen (20 minutes)

were applied for immunohistochemical analysis of
antibody binding. Non-immune mouse serum
served as a negative control, and Mayer’s hematoxylin was used as counterstain. Cytoplasmic
staining was considered evidence of positivity.
An expert pathologist reviewed the slides. In
each slide, to evaluate the inflammatory cells,­initially hematoxylin–eosin (H-E) sections wereprepared and examined under light microscopy.
For each H-E section, we identified inflamma­tory
cells: polymorphonuclear cells (PMNCs), including
polymorphonuclear leukocytes and eosinophils;mononuclear cells (MNCs), including lympho­
cytes, plasma cells, mast cells, and histiocytes; andfibroblasts. Then iNOS expression and number of
inflammatory cells were determined under lightmicroscopy­ (Leica, Germany) per 100 cells in 3–4
high-magnification fields. Means of cell counts
were calculated as % values, and iNOS PI values
for inflammatory cells were evaluated on a scale of
0 to 3: PI 0, antigen (iNOS)+ cell count was 0% (nostained cells); PI 1, antigen (iNOS)+ cell count was<5%; PI 2, antigen (iNOS)+ cell count was 5–50%;
and PI 3, antigen (iNOS)+ cell count was >50%.
PI. For the quantitative assessment of iNOS expression, staining in the E, SE, and D and inflammatory cells was assessed by counting 100 cells in
3–4 high-magnification fields and calculatingmeans. Means for the iNOS (+) cells per 100 cells
on a high-magnification field (×400) were derived
in the E, SE, and D. Scoring was performed on a
0–3 scale, in which 0 represented negative staining;
1, weakly positive; 2, positive; and 3, strongly posi­
tive.12 For the PI, values indicated the following: PI
0, antigen (iNOS)+ cell count was 0% (no stained
cells); PI 1, antigen (iNOS)+ cell count was <5%;
PI 2, antigen (iNOS)+ cell count was 5–50%; andPI 3, antigen (iNOS)+ cell count was >50%.
Assessment was performed at eight levels: (1)Epithelial_apical (EP_apical), (2) Epithelial_basal(EP_basal), (3) Subepithelial_perivascular (SE_pv),(4) Subepithelial_glandular (SE_gland), (5) Subepithelial_ endothelial (SE_endothelial), (6) Deep_
perivascular (D_pv), (7) Deep_glandular (D_gland), and (8) Deep_Endothelial (D_endothelial).
Statistical analysis
The statistical packet for SPSS (version 16.0) wasused for statistical evaluation. For the four groups– nasal cavity, maxillary sinus, ethmoid sinus, and210

N. Bayar Muluk et al.
sinus, and nasal cavity and control group samples,and for MNC-% and iNOS-PI, PMNC-%, and­iNOS-PI, and all-cell iNOS-PI.
For iNOS_D_gland PI, the four groups differedsignificantly (p = 0.043), but no statistically significant difference was found for other iNOS PI valuesof the mucosal layers (p>0.05) (Table 1). To identify the group responsible for the significant dif­ference in the iNOS_D_gland PI, pairwise comparisons were performed with Bonferroni correction,
but no significant difference was detected(p>0.0125) (In Bonferroni adjustment process,­adjusted alpha value was used. alphaadjusted
value= 0.0125).
­
A statistically significant difference was foundfor PMNC-% (p = 0.011) and iNOS-all_cells(p = 0.043) among the four groups (Table 1). To
identify the group responsible for the difference,
pairwise comparisons were performed with Bonferroni correction and showed that PMNC-% values for the ethmoid sinus (median 25.0) (p = 0.005)
and maxillary sinus (median 55.0) (p = 0.006) weresignificantly higher than for the control group (median 5.0) (Figure 2). In addition, the iNOS-PI of allcells of the ethmoid sinus (median 2.0) was significantly higher than that of the control group (median1.0) (p = 0.010) (Figure 3).

control – the differences for iNOS PI were analyzed
using Kruskal-Wallis variance analysis. When astatistically significant result was found, pairwise
comparisons were made using the Mann-Whitney
U Test with Bonferroni correction to detect thegroup that caused the difference.
In the study group, correlations between age,sex, polyp duration, smoking, and Brinkmann index values and iNOS levels in the ethmoid sinus,
maxillary sinus, and nasal cavity were respectively
analyzed separately using Spearman’s correlationrho efficient in the three polyp groups.
The confounding factors (covariates: age, sex,polyp duration, smoking, and Brinkmann index,
PMNC-%, MNC-%, iNOS-PMNC, iNOS-MNC,
iNOS-all cells) affecting iNOS-PI in mucosal ­layers(E, SE, and D) were analyzed using linear regression analysis (backward, step-by-step analysis) inthe three polyp groups. A p value <0.05 was considered statistically significant.
Results
Table 1 and Figures 1-3 illustrate medians of iNOS
PI values in the EP_apical, EP_basal, SE_pv, SE_
gland, SE_endothelial, D_pv, D_gland, and D_­endothelial parts of the ethmoid sinus, maxillary

Table 1iNOS positivity index levels in ethmoid sinus, maxillary sinus, nasal cavity, and control groups (medians)
iNOS positivity index
iNOS_EP_apicaliNOS_EP_basal

iNOS_SE_pv

iNOS_SE_glandiNOS_SE_endothelial

iNOS_D_pv

iNOS_D_gland

Ethmoid sinus(n = 14)
2.0

2.0

1.0

2.0

1.0

1.0

1.0

GroupsMaxillary sinus(n = 10)
2.0

2.0

1.5

2.0

1.0

1.0

1.5

Nasal cavity(n = 10)
2.0

2.0

1.0

2.0

0.5

1.0

0.5

Control(n = 9)
2.0

2.0

1.0

2.0

1.0

1.0

2.0

p*
0.502

0.116

0.480

0.223

0.434

0.631

0.043

iNOS_D_endothelial

1.0

1.0

0.0

0.0

0.055

PMNC-%

25.0

25.0

22.5

5.0

0.011

MNC-%

75.0

77.5

90.0

0.423

iNOS-all_cells

2.0

2.0

1.0

Inflammatory cells
iNOS-PMNCiNOS-MNC

0.5

2.0*p value shows the results of Kruskal–Wallis variance analysis.

0.0

2.0

0.0

2.0

0.463

0.228

0.043

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iNOS in sinonasal polyp pathogenesis

Figure 1
iNOS positivity index levels in mucosa of groups 1–4

In the polyp group, correlations between age,sex, polyp duration, smoking, and Brinkmann index values and iNOS levels in the ethmoid sinus,
maxillary sinus, and nasal cavity were analyzed
separately. For the ethmoid sinus, as polyp duration
increased, iNOS_D_pv decreased (p = 0.017,
r = –0.727). In patients with a higher Brinkmann
index, iNOS_D_endothelial decreased (p = 0.037,
r = –0.633). For the maxillary sinus, as age increased, iNOS_SE_pv also increased (p = 0.027,
r = 0.691), and iNOS_D_pv increased with longer
polyp duration (p = 0.028, r = 0.808).
The confounding factors (covariates: age, sex,polyp duration, smoking, and Brinkmann index,
PMNC-%, MNC-%, iNOS-PMNC, iNOS-MNC,
iNOS-all cells) affecting iNOS-PI in the layers ofthe mucosa (epithelial, subepithelial and deep)
were analyzed by linear regression analysis (Figure4). For iNOS_EP_apical, as iNOS-PMNC PI increased, iNOS_EP_apical decreased (p = 0.038,
r = –426) while for the iNOS_SE_gland, as PMNC% increased, iNOS_SE_gland value decreased(p = 0.017, beta = –6.602), as it also did with increasing MNC-% (p = 0.020, beta = –6.348).
iNOS_D_pv, on the other hand, was increased insmoking patients (p = 0.032, r = 0.745) but decreased in patients with a higher Brinkmann index(p = 0.012, r = –0.896). Finally, in the ethmoid andmaxillary sinuses, iNOS_D_endothelial values increased relative to nasal cavity; whereas they decreased in the nasal cavity relative to ethmoid andmaxillary sinuses (Median values of iNOS_D_endothelial values were 1.0, 1.0 and 0.0 in ethmoid
sinus, maxillary sinus and nasal cavity polyps respectively) (p = 0.014, r = –0.532) (Figure 4).
Histopathologic findings
Light microscopy examination revealed that pseudostratified ciliated epithelium was present in the
majority of polyps, and a very small portion of thepolyps were also lined with metaplastic epithelium.
In iNOS-stained sections, strong positivity was
specifically detected in the apical portion of the212

N. Bayar Muluk et al.

Figure 2
Distribution of inflammatory cells in the mucosa.
Based on the Mann–Whitney U test with Bonferroni correction, PMNC-% values of ethmoid sinus (median 25.0) (p = 0.005) andmaxillary sinus (median 55.0) (p = 0.006) were significantly higher than that of the control group (median 5.0).

Figure 3
iNOS PI levels in inflammatory cells.
Based on the Mann–Whitney U test with Bonferroni correction, iNOS-PI of all cells of ethmoid sinus (median 2.0) was significantly
higher than that of the control group (median 1.0) (p = 0.010).

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iNOS in sinonasal polyp pathogenesis

Figure 4
Coefficient (beta) values and covariates as significantly detected confounding factors on iNOS PI, as detected by linear regression
analysis (backward).
(–) Coefficient (beta) values are presented. Covariates were age, sex, polyp duration, smoking, and Brinkmann index, PMNC-%,
MNC-%, iNOS-PMNC, iNOS-MNC, and iNOS-all cells. When the iNOS-PMNC PI increased, iNOS_EP_apical decreased (p = 0.038,
r = –426); as PMNC-% increased, iNOS_SE_gland value decreased (p = 0.017, beta = –6.602); as MNC-% increased, iNOS_SE_
gland value decreased (p = 0.020, beta = –6.348); in smoking patients, iNOS_D_pv value increased (p = 0.032, r = 0.745); in patientswith a higher Brinkmann index, iNOS_D_pv value decreased compared to patients with lower Brinkmann Index (p = 0.012, r = –0.896);
and in ethmoid and maxillary sinuses, iNOS_D_endothelial values increased whereas in the nasal cavity, iNOS_D_endothelial values
decreased (p = 0.014, r = –0.532).

s­ urface epithelium. In subepithelial areas, both themucosal gland epithelium and endothelium showed
diffuse immunostaining with iNOS. Although
MNCs and fibroblasts showed prominent iNOS
­expression, except for a slight expression in the­ethmoid sinus, almost no expression could be ­detectedin polymorphonuclear leukocytes. In edematousa­ reas of polyps, expression of all markers was lessthan in other areas (Figure 5).
Discussion
Nasal polyps is a multifactorial disease with infectious, noninfectious, inflammatory, anatomic, and214

Figure 5
Immunohistochemical staining for iNOS in a polyp sample
displaying expression in both the epithelial apical and basal
layers. In the subepithelial layer, diffuse positive staining wasobserved in MNCs, vascular endothelial cells, and glands(×100).

genetic abnormalities. Chronic inflammation remains the central major factor in nasal polyps.2­Endogenously produced anti-microbial peptides
like beta-defensins and molecules like NO are­important components of the immune response.
Disturbance of this response can have damaging
consequences. Nasal polyposis, a common disorder, is an example of such immune dysregulation.13
NO concentrations in healthy sinuses can riseclose to the highest permissible environmental pollution levels, leading to the hypothesis that NO
contributes to sinus host defense by virtue of its antimicrobial properties and ability to upregulate ciliary activity.14 The most important source of NO in
the respiratory tract is iNOS. In the upper and lower
airways, iNOS is primarily expressed in epithelial
cells and macrophages and is upregulated by cytokines, microbes, or microbial products, permitting
NO production to increase markedly in response toinfection and in inflammatory states.14
Lorente et al.15 reported that nasal polyps containhigher levels of total NO synthase activity than nasal mucosa tissue. In addition, nasal polyps exhibit
mainly iNOS activity whereas all NOS activity detected in the nasal mucosa is in constitutive form. In
both cases, NOS activity localizes in epithelial
cells. In view of these findings, we inferred that NO
may be an important inflammatory mediator in the

N. Bayar Muluk et al.
respiratory system and that the epithelium may be asource of NO production in the upper airways.16
In the present study, we investigated the role ofiNOS in the pathogenesis of nasal polyps. No statistically significant difference for iNOS PI values
was found in the mucosal layers among the fourgroups. iNOS positivity was decreased from the SE
to D layers because of stromal edema and reduced
cell density. In other words, in edematous areas ofpolyps, expression of all markers was less than inother areas, which we attribute to the small numberof cells in the edematous areas. This condition may
explain the lack of difference in results of the statistical analysis of the four groups.
Strong eNOS immunoreactivity and weak iNOS
immunoreactivity have been found in the columnar
epithelium of human nasal mucosa in normal subjects.17 In our study, however, intense iNOS expression was detected at the apical surface of the epithelial layer in all groups. Also, in our study, thepercentage of PMNCs in the ethmoid and maxillarysinuses and iNOS-PI values of all cells of the­ethmoid sinus were significantly higher comparedto the control group. NO levels may reflect the­inflammatory status of both the upper and lower
airways. Because NO synthase is induced in inflamed upper airways,16 an increase in inflammatory
cells expressing iNOS in the ethmoid and maxillarysinuses was thought to play a role in the patho­
genesis of sinonasal polyps in our study.
In addition, we observed that iNOS was not expressed in PMNCs except for very low expression
in the ethmoid sinus. In the all-cells group of inflammatory cells, there were PMNCs, MNCs, andfibroblasts. Higher iNOS expression in the all-cells
group compared to MNCs was the result of fibroblasts and their higher iNOS expression. Thus,
iNOS expression in the all-cells group was mainly
due to fibroblastic iNOS expression.
In addition to a role in nonspecific immunoreactions and inflammation in a variety of tissues, NO
can play an important role in the regulation of­vascular tone and neurotransmission. Many kindsof cells, such as macrophages, neutrophils, and­endothelial and smooth muscle cells can express
iNOS. Because all isoforms of NOS seem to bepresent in nasal tissues and the expression of iNOS
under inflammatory conditions seems to be responsible for excessive production of NO, the distribution of NOS isoforms (especially iNOS) in normal
and inflammatory nasal tissue, as well as the exact

iNOS in sinonasal polyp pathogenesis
requirements for expression of iNOS, remains to beshown.18 In the present study, at least, in patientswith longer polyp duration and D-pv and in patientswith a higher Brinkmann index value, D-endothelial iNOS PIs in ethmoid sinus and D-pv iNOS expression decreased. However, in maxillary sinus inolder patients and in patients with longer polyp duration, perivascular iNOS PIs increased in the subepithelial and deep layers of the mucosa, respectively. Lower iNOS-endothelial PI levels may affectthe vascular structure of the polyps, lessening circu­
lation and resulting in more fibrotic polyps. Longterm and excessive smoking were also related
to lower iNOS values at the perivascular region,
causing a reduction in vascular permeability andless migration and extravasation of inflammatory
cells to the mucosal stroma. Ultimately, the inflammatory reaction lessens and fibrotic polyps emerge.
In older patients with longer polyp duration, iNOSperivascular PI values increased in the deep layerof the mucosa, causing increased vascular permeability and stromal edema.
Hess et al.,18 working with non-inflamed fresh
human nasal mucosa from the middle turbinate,compared these samples immunohistologicallywith nasal mucosa manifesting the typical findingsof chronic polypoid rhinosinusitis (i.e., polypoid
middle turbinates and polyps of the middle nasalduct). Clearly increased or initiated expression of
iNOS was detected in blood vessels, glands, macrophages, and epithelium of chronically inflamed andbacterial lipopolysaccharide-incubated nasal tissue
in comparison to the non-inflamed controls.18 In ourstudy, increased PMNC percentages and iNOS PI
values were related to decreased iNOS expression
in the EP_apical and SE_glandular portions. Increased MNC percentages were also seen with lower
iNOS expression in the SE_glandular area. The­results suggest that the increase in both PMNCs and
MNCs was related to a decrease in iNOS glandular
PI values and that fibroblasts, the other cells of the
all-cells group (PMNCs+MNCs+fibrobasts) may
be the main source of iNOS values in polyp tissues.
Tewfik et al.19 reported that NO stimulates collagen
expression in human nasal polyp–derived fibroblasts. This stimulation appeared to favor the upregulation of collagen type III, leading to a shift in
the ratio of collagen type I to type III production.19
In the deep endothelial parts of our polyp tissue
samples, we observed increased iNOS expression
in the ethmoid and maxillary sinuses and decreased

215
iNOS expression in the nasal cavity. iNOS is induced by low oxygen tension.20 At low tissue oxygen levels in nasal polyps, iNOS values increase inendothelial cells, causing increased vascular permeability. Vasodilatation and inflammatory cell
migration may initiate the process of polypoid
degeneration­ and edema in the stroma. Liu et al.21
examined the expression and distribution of
hypoxia­-inducible factor-1alpha in nasal polyps
and concluded that hypoxia may play an importantrole in the pathogenetic mechanism of nasal polyps.
In our study, iNOS expression was detected inboth polyp tissues and the inferior turbinate samples of the control group, although with a significant difference among the four groups. In polyptissue, because of edema in the subepithelial anddeep layers of the mucosa, the cellular density was
not as intense in these areas compared to controls.
As noted, iNOS expression occurs in many kinds ofcells,22 which may explain the lack of differencesamong iNOS expression values. Watanabe and­Kakuta23 studied iNOS expression under light andelectron microscopy, and immunoreactivity for
iNOS was reported to be localized to the mucosal
epithelium, inflammatory cells, vascular endothelium, smooth muscle, and nasal gland.23
Like Kirtsreesakul,2 we suggest that chronic inflammation remains the central major factor in nasal polyps. Fundová et al.,24 investigating the expression of insulin-like growth factor-1 receptor(IGF-1R) and iNOS in nasal polyps compared tohealthy nasal mucosa in both the epithelial and stromal compartments, detected positivity for iNOS
within the epithelium of nasal polyps comparedwith the nasal mucosa. The numbers of iNOS-positive single cells were highly increased in nasal
polyp­ vs. nasal mucosa in both epithelial (3.83 vs.
1.08) and stromal (4.96 vs. 2.67) compartments. An
increased iNOS expression within the epithelial
layer as well as increased number of iNOS- andIGF-1R–positive cells in nasal polyps also was observed. Their results suggest that the innate immune
mechanism and to a lesser extent growth and­homeostasis of epithelial cells may play a role information of nasal polyps. Chen and Xiang25 also
reported that iNOS was expressed mostly in the­inflammatory cells inside the tissue; a large amountof NO produced by iNOS may lessen the inflammation process of nasal polyps.
The cellular expression of NOS in the human­nasal mucosa suggests a possible role for iNOS in

216
the regulation of blood flow, nasal secretion, andciliary movement in health and disease.7 In ourstudy, we inferred that iNOS may potentially play arole in sinonasal polyp pathogenesis through thevascular system, endothelial cells, and glandular
cells, especially in the subepithelial and deep layersof the mucosa. Increased vascular permeability,
stromal edema, inflammatory cell migration into
the stroma of the mucosa, and increased secretionby mucosal glands may result in polyp formation.
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Dr. Nuray Bayar Muluk
Birlik Mahallesi, Zirvekent 2. Etap Sitesi, C-3 blok, No: 62/43
06610 Çankaya, Ankara, Turkey
Tel.: +90 312 4964073, +90 532 7182441
Fax: +90 318 2252819
E-mail: nurayb@hotmail.com
E-mail: nbayarmuluk@yahoo.com