Role of allergy and mucosal inflammation in nasal polyps and chronic sinusitis 2

2.5 Inflammatory Cell Scores in Nasal Polyps and Chronic Sinusitis 2.5.1 Inflammatory Cell Scores in Nasal Polyps and Its Paired Middle Turbinate, and Middle Turbinate of Allergic Rhini

Chapter 2 Inflammatory Cell Patterns in Nasal Polyps and Chronic Sinusitis2.1 Introduction

Nasal polyps and chronic sinusitis are common related diseases worldwide In

chapter 1, the epidemiology and pathogenesis of these diseases was briefly reviewed

They are multifactorial diseases closely related with asthma,1,2 aspirin intolerance,3

cystic fibrosis,4 rhinitis and especially nonallergic rhinitis,5,6 immunodeficiency,7

primary ciliary dyskinesia,8,9 and other underlying diseases Although many theories

have been suggested, the roles of allergy and infection remain the most important and

controversial underlying mechanisms in nasal polyp and chronic sinusitis In addition,

although the two diseases are concomitant in many patients, the pathogenic

mechanism interlinking these two common nasal diseases is still incompletely

understood

Most of the studies in nasal polyp and chronic sinusitis were carried out in Caucasians

In Asia, data on their etiology and pathophysiology are still lacking Previous studies

suggested that the respective epidemiology and aspects may differ in the Caucasian

and Asian populations The incidence rate of nasal polyps in Caucasians was reported

to be 1% to 4.3%.10-14 The estimated prevalence of sinusitis in Europe and US ranges

from 10% to 40%.15-17 However, in a national survey in Korea reported by Min et

al.,18 the incidence rate of nasal polyps and chronic sinusitis was 0.5% and 1.01%,

respectively Etiology factors may also play various roles in different populations For

example, cystic fibrosis, a common associated disease with nasal polyps and chronic

sinusitis, is rare in Asian populations and the use of aspirin is also not as common as

that in western countries It was also suggested that chronic sinusitis in Asian

populations had a higher incidence rate of nasal polyps than in Caucasians due to the

narrower nasal passages.19 A difference in the pathogenesis of nasal polyps between

Caucasians and Asians has also been suggested As introduced in chapter 1, nasal

polyps can be differentiated into four subgroups according to histophathology.20

Eosinophilic and neutrophilic nasal polyps are the two major subtypes which account

for 85%-90% and 10% of the cases, respectively, in Caucasians.20 In Asians, a

relatively higher incidence of neutrophilia (40%) and a relatively lower incidence of

eosinophilia (41.7% to 65.2%) in nasal polyps have been suggested.21,22 However, a

recent study by Lacroix et al.23 showed no major histological difference in nasal

mucosa and polyp tissues obtained from African, Chinese and Caucasian patients

2.2 Aim of Study

2.2.1 Hypothesis

The underlying mechanism of chronic sinusitis and nasal polyps involves a complex

inflammation characterized by infiltration and activation of various types of

inflammatory cells In this study, it is proposed that:

I The cell pattern and role of inflammatory cells in these diseases may differ depending on local and systemic triggering factors, i.e., allergy and infection

II There exists an auxiliary effect between the unaffected and diseased nasal

mucosa in terms of inflammatory cells and cytokines leading to pathogenesis of the

excised nasal polyps This will help to explain the recurrent nature of nasal polyps

III Not only eosinophils but also neutrophils and lymphocytes may play important

roles in the persistence of mucosal inflammation and in inducing nasal airway

remodeling

IV There may be ethnic based differences controlling cell patterns involved in the

chronic inflammation of nasal polyps and chronic sinusitis

2.2.2 Specific Aims

The specific aims of this study are:

I To study the type of cellular mechanisms and local tissue immune response in

affected sinus mucosa and nasal polyp tissue using an immunohistochemical

characterization of inflammatory cells and comparing these findings with that of

normal nasal mucosa

II To compare the spatial distribution of inflammatory cells in affected sinus

mucosa/polyps with the biopsy specimens obtained from the uninvolved middle

turbinate at the same site The purpose of this comparison is to study the nature and

localization of mucosal inflammation and the possibility of interactions between the

two sites

III To explore the relationship between the inflammatory cell pattern in sinus

mucosa/polyps and clinical hypersensitivity and underlying diseases

IV To explore the ethnic based difference of chronic inflammation in nasal polyps

and chronic sinusitis by comparing our results for local patients with those reported

for Caucasian patients

This study allows for a better understanding of the pathogenic mechanisms of chronic

sinusitis and nasal polyps The results of this study will aid in the discovery of

better-targeted therapies and preventive measures In addition, the exploration of

ethnic differences may suggest the contribution of genetic predisposition or

Patients who present with following symptoms for 12 weeks or more: anterior and/or

posterior mucopurulent drainage and nasal congestion Nasal endoscopic examination

shows discolored mucus or edema of the middle meatus or ethmoid area Furthermore,

a positive sinus CT scan with confirmation of mucosal disease is required

II Nasal polyps

Patients may have symptoms like stuffy nose, difficulty smelling odors and/or facial

pain Nasal endoscopic examination shows pale, semitranslucent, watery masses

protruding into the nasal cavity CT scan is used to determine the condition in

paranasal sinuses

III Allergic rhinitis

The occurrence of two or more symptoms (nasal obstruction, rhinorrea, sneezing and

itchy nose) on most days during the past year If patients coexisted with atopy, allergic

rhinitis is diagnosed

IV Atopy

A positive serum specific IgE (equal or more than 0.35 IU/ml) to at least one of the

inhalant allergens tested

2.3.2 Study patients

In this prospective study, patients with nasal polyps and chronic sinusitis, allergic

rhinitis and non-atopic, non-rhinitis controls were randomly selected from the

department of Otolaryngology, Head & Neck Surgery of the National University

Hospital of Singapore as follows:

I Forty-eight patients, 34 males and 14 females, aged from 12 to 78 years (mean

age 44) with unilateral/bilateral nasal polyps, who were scheduled for functional

endoscopic sinus surgery The diagnosis of nasal polyps was based on medical history

and clinical examinations, including nasal endoscopic examination and CT scan

Among the above nasal polyp patients, six patients had available nasal polyp tissue,

sinus mucosa as well as the paired middle turbinate All of them were diagnosed as

having nasal polyps with concomitant chronic sinusitis They were four males and two

females, aged from 28 to 51 years (mean age 43) This small group was used for the

exploration of any possible correlations between chronic sinusitis and nasal polyps

II Twenty patients, ten males and ten females, aged from 19 to 76 years (mean age

47) with unilateral/bilateral chronic sinusitis but no nasal polyps, who were scheduled

for functional endoscopic sinus surgery in our department The diagnosis of chronic

sinusitis was based on medical history and clinical examinations, including nasal

endoscopic examination and CT scan

III Fifteen patients, 14 males and one female, aged from 19 to 62 years (mean age 27)

with allergic rhinitis who were scheduled for septal surgery in our department Their

atopy status was proved by the ImmunoCAP system (Pharmacia Diagnostics, Clayton,

NC) These patients had no history of chronic sinusitis or nasal polyps

IV A control group of fourteen non-rhinitis, non-atopic patients, 11 males and 3 females, aged from 22 to 39 years (mean age 27), with septal deviation who were

scheduled for septal plastic surgery Patients with nasal polyps, sinusitis, allergic

rhinitis and atopy were excluded

All subjects were specifically asked for a history of aspirin exposure and asthma

Patients with a history of paroxysmal attacks of breathlessness commonly associated

with a tightness of the chest and wheezing were referred to the respiratory physician

for further evaluation of asthma All patients had a trial of an intranasal

glucocorticosteroid spray but did not show a relief of their symptoms Their

medication was discontinued for more than one month prior to surgery.24,25 A signed

informed consent was obtained from the study patients before surgery Approval to

conduct this study was granted by the National Medical Research Council of

Singapore and the institutional review board of the Medical Faculty of the National

University of Singapore

Table 8 Patient groups in the study of inflammatory cell pattern

Patient group Mean age Number of patients Male/Female

A pair of biopsies was taken, one from the nasal polyp/inflamed sinus mucosa and the

other from the ipsilateral middle turbinate One biopsy sample was taken from the

middle turbinate of allergic rhinitis and control patients during septal plastic surgery

Specimens were embedded in a tissue-freezing medium (Leica Instruments GmbH) in

liquid nitrogen immediately after resection The frozen samples were kept at -80°C for

further study

2.3.3.2 Immunohistochemistry Staining 26

Sections of 4 µm were prepared in a cryostat and attached onto gelatinized slides and

allowed to dry at room temperature overnight The sections were fixed in pure acetone

for 10 minutes at 4°C followed by washing with PBS-TX (Phosphate-buffered saline

with 0.1% Triton X-100, pH 7.4) 3 times, for 10 minutes each The slides were then

incubated in 0.3% H2O2 for 30 minutes at room temperature to reduce nonspecific

background staining due to endogenous peroxidase After washing with PBS-TX

again, the slides were incubated with 5% normal goat serum for 1 hour at room

temperature Mouse anti-human monoclonal antibodies (Table 9) with appropriate

dilution (1:200 to 1:100) were incubated overnight at 4°C for immunohistochemical

staining The next day, the slides were washed with PBS-TX and incubated with

secondary antibody (BD Biosciences Parmingen, biotinylated polyclonal goat

anti-mouse Ig with dilution of 1:300 in PBS) at room temperature for 1 hour After

washing, ABC (avidin-biotin complex, DakoCytomation) was applied and incubated

for 1 hour at room temperature followed by another washing in PBS-TX DAB

(diaminobenzidine tetrahydrochloride, DakoCytomation) was used for color

development for 5-10 minutes After rinsing with distilled water, the sections were

counterstained with Mayer’s hematoxylin (Sigma-Aldrich Corporate) for a further 5

seconds, dehydrated with series ethanol (90%, 100%, 100%), and cleared with xylene

(3 times), and mounted with a DePeX mounting medium (BDH Laboratory supplies)

Control staining for nonspecific staining was routinely performed with PBS instead of

primary antibodies, and all trials proved negative To test the specificity of anti-CD4+

(helper T cells) and anti-CD8+ (cytotoxic/suppressor T cells), fresh human tonsil

specimens were obtained Consecutive samples were stained separately with anti-CD4,

anti-CD8 and anti-CD3 antibodies (Lab Vision NeoMarker, Rabbit anti-human

monoclonal CD3, clone SP7) with the same protocol as mentioned above The sum of

CD4+ and CD8+ cells was approximated to the number of CD3+ cells

Table 9 Mouse anti-human monoclonal antibodies used

Anti- CD4 DakoCytomation MT310 Helper/inducer T cells and

subpopulation of macrophages Anti-CD8 DakoCytomation C8/114B Suppressor/cytotoxic T cells

Anti-CD19 DakoCytomation HD37 Precursor and mature B cells

(no plasma cells)

Neutrophil elastase DakoCytomation NP57 Neutrophil

Major basic protein BD Biosciences

Parmingen

AHE-2 Eosinophil

2.3.3.3 Cell Counting

Three areas with high intensity positive cell distribution were selected in each section

and a cell count was performed under a light microscope at magnification of 400 The

positive cells stained with peroxidase-labeled monoclonal antibody on cell

membranes were counted Cell counting was averaged and evaluated with scores from

0 to 3.27,28 The counting was performed blindly without knowing the identity of the

samples:

0: no positive staining cells;

1 (+): A few (1-10) positive cells;

2 (++): A moderate number (11-20) of positive cells and some cluster of positive cells;

3 (+++): Many (>20) positive cells

For CD4+ and CD8+ cells, additional absolute cell counts were performed The mean

numbers of cell counts in three power fields were calculated

Level of Allergen Specific

Three milliliters of peripheral blood was taken during surgery Serum total IgE (tIgE)

and specific IgE (sIgE) to a common panel of inhalant allergens, including

Dermatophagoides pteronyssinus, Dermatophagoides farinae, Aspergillus fumigatus,

cockroach, common pollen and ragweed mixtures (Bermuda grass, Ambrosia

artemisiifolia, Ambrosia elatior) were determined using the ImmunoCAP system

Patients with sIgE ≥0.35 IU/ml to at least one of the testing allergens were considered

as atopy sIgE was classified into seven scores shown in Table 10

2.3.5 Statistical Analysis

A standard personal computer with SPSS (Statistical Package for the Social Sciences)

11.5 software (SPSS, Inc., Chicago, Illinois, US) was used for the statistical

evaluation of the results According to the character of data, the appropriate method

was applied

2.3.5.1 Cell Score Analysis

I To analyze the correlation of inflammatory cell infiltration in one sample,

nonparametric Spearman’s correlation test was used

II To compare the distribution of inflammatory cell expression within the groups

(nasal polyp/inflamed sinus mucosa and paired middle turbinate from the same side),

Wilcoxon signed rank test for 2-related samples was used

III To explore the correlation of inflammatory cell infiltration in nasal

polyp/inflamed sinus mucosa and its paired middle turbinate, nonparametric

Spearman’s correlation test was used

IV To compare the cell distribution between different groups (between nasal

polyp/sinusitis patients with and without atopy; between nasal polyp/sinusitis patients

with and without asthma; between nasal polyp/sinusitis patients and controls),

Wilcoxon rank sum test for 2-independent samples was applied

V In the subgroup of patients having nasal polyps and chronic sinusitis with available nasal polyp tissue, sinus mucosa as well as middle turbinate mucosa from

the same side, Friedman test was used to test the distribution difference of

inflammatory cells in the three samples Kendall's W test with Kendall's W coefficient

of concordance was applied to test any possible correlation among the three samples

2.3.5.2 Exact Count of CD4+ and CD8+ T cells

I To test the normality of cell counting, one-sample Kolmogorov-Smirnov Test was

used

II To test the distribution of T cells between paired samples, Wilcoxon Signed

Ranks test was applied

III To test the correlation of T cells between paired samples, Pearson’s correlation

was used

IV To test the distribution of T cells between different groups,

Kolmogorov-Smirnov test for different distributions was applied

2.3.5.3 Correlation of Inflammatory Cells with tIgE and sIgE

I To explore the correlation between tIgE and inflammatory cell score, nonparametric Spearman’s correlation was applied

II To explore the correlation between inflammatory cell score and sIgE (score),

nonparametric Spearman’s correlation was applied

2.3.5.4 tIgE and sIgE in Different Study Groups

I To compare tIgE in different groups, Kolmogorov-Smirnov test for

2-independent samples was applied

II To compare sIgE in different groups, Wilcoxon rank sum test for 2-independent samples was applied

In all the tests, a P value of less than 0.05 was regarded as significant For the

correlation analysis, a correlation coefficient above 0 is taken as positive correlation;

0-0.3 as a weak correlation, 0.3-0.5 as a medium correlation, and a strong correlation

of over 0.5

2.4 Histology, Etiology and Serum IgE

2.4.1 Quality Control Staining for CD4+ and CD8+ T Cells

Figure 7 Quality controls of anti-CD4

and anti-CD8 antibodies staining in tonsils under light microscope 100× A Anti-CD3 B Anti-CD4 C Anti-CD8

C Anti-CD8

Quality control of anti-CD4 and anti-CD8 antibodies performed in tonsils proved that

the sum of CD4+ cells and CD8+ cells was almost the same as the number of CD3+

cells (Figure 7) CD4+ T cells are prominent over CD8+ T cells in tonsils

Figure 8 Histological changes in nasal polyp/inflamed sinus mucosa (under 100× light

microscope except C.) A Nasal polyp tissue has edema with high infiltration of inflammatory cells Epithelium shows severe basal cell hyperplasia Glands totally disappear B Nasal polyp

tissue with high edema, infiltration of inflammatory cells, disappearance of glands and hyperplasia

of goblet cells C Nasal polyp tissue (same as B.) under 200× light microscope Goblet cell hyperplasia is shown D Nasal polyp with high edema Glands disappear totally Epithelium is

damaged (pink arrow) Basement membrane thickened (red arrow)

As introduced in chapter 1, nasal mucosa has a typical airway structure which is

characterized by a pseudostratified columnar ciliated mucus membrane with goblet

cells and metaplastic squamous cells Underlying it is the basement membrane and

loose connective tissue which contains blood vessels, submucosal glands and other

cells The typical changes in the histopathology of nasal polyps and inflamed sinus

mucosa were quite similar in our study of patients, including: infiltration of

inflammatory cells, which will be discussed later; structural changes of epithelium

including hyperplasia of basal cells and goblet cells, and metaplasis of squamous cells;

structure change of glands; basement membrane thickening; and edema Figure 8

shows the typical histology changes in nasal polyp tissue These changes are also

representative of the pathological changes in the inflamed sinus mucosa of chronic

sinusitis patients

2.4.3 Etiology of Nasal Polyps

2.4.3.1 Etiology Factors

In the nasal polyp group, the ethnic classes were 38 Chinese, two Malays, five Indians,

one Philippine, one Nepalese and one British Unilateral and bilateral nasal polyps

were shown in 11 and 37 patients, respectively Forty-seven patients had sinusitis

(98%), seven unilateral and 40 bilateral, as was confirmed by CT scan of the sinuses

Only one patient had a unilateral polyp and did not show clinical signs of sinusitis

(including in the CT scan) Four patients (8%) had concomitant asthma that was

diagnosed by respiratory physicians Only one patient (Indian) had been previously

diagnosed with cystic fibrosis by a sweat test and genotyping in Australia None of the

study patients had shown a history of aspirin intolerance The male/female ratio was

about 2.4 In the allergic rhinitis group, there was only one patient with asthma In the

control group, no history of asthma was evidenced

2.4.3.2 Atopy and tIgE Measurements

Table 11 shows the results of atopy and tIgE measurements Sera were available for

allergy testing from 37 patients with nasal polyps, and all the subjects in the allergic

rhinitis group and controls Atopy and high levels of serum tIgE (tIgE≥100 IU/ml) were evidenced in 11 (29.7%) and 14 (38.9%) patients with nasal polyps, respectively

Only one nasal polyp patient showed atopy with serum tIgE less than 100 IU/ml All

of the allergic rhinitis patients had proved to be atopic by a serum sIgE test 13

(86.7%) of them had high levels of serum tIgE None of the control subjects showed

atopy or high serum tIgE

Table 11 Incidence rate of atopy and tIgE levels in patients with nasal polyps (n=37),

allergic rhinitis (n=15) and controls (n=14)

Nasal Polyp with/without sinusitis

(n=37)

11 (29.7%)

14 (38.9%)

34 (61.1%) Allergic rhinitis

(n=15)

15 (100%)

13 (86.7%)

2 (13.3%) Controls

(n=14)

0 0 14

(100%)

*Presence of serum sIgE≥ 0.35 IU/ml to at least one of the common allergens tested

Only one nasal polyp patient was found to be atopic in the group of tIgE<100 IU/ml.

tIgE in nasal polyp patients, allergic rhinitis patients and controls was compared by

Kolmogorov-Smirnov test for two independent samples Allergic rhinitis patients had

significantly higher serum tIgE than nasal polyp patients and controls There was no

significant difference between nasal polyp patients and controls

According to Wilcoxon rank sum test for 2-independent samples, allergic rhinitis

patients had significantly higher scores of sIgE to Dermatophagoides pteronyssinus

and Dermatophagoides farinae than nasal polyp patients and controls Allergic rhinitis

patients also showed significantly higher sIgE to cockroach than nasal polyp patients

There was no significant difference in sIgE to common allergens tested between nasal

polyp patients and controls

2.4.4 Etiology of Chronic Sinusitis

2.4.4.1 Etiology Factors

The ethnic classification of the chronic sinusitis patients composed 15 Chinese, four

Malays and one Indian Unilateral and bilateral chronic sinusitis was shown in 11 and

9 patients, respectively Two patients (10%) had concomitant asthma with bilateral

chronic sinusitis None of the study patients had shown a history of aspirin intolerance

or cystic fibrosis The ratio of male/female was 1.2

2.4.4.2 Atopy and tIgE Measurements

16 chronic sinusitis patients had sera available for allergy test Atopy and high serum

tIgE (tIgE≥100 IU/ml) were evidenced in 6 (37.5%) and 9 (56.3%) of the patients respectively All the patients with atopy had a high level of tIgE while three patients

(19%) with tIgE over 100 IU/ml did not show atopy

There was no significant difference in the tIgE levels between chronic sinusitis

patients and allergic rhinitis patients/controls Allergic rhinitis patients had a

significantly higher level of sIgE to Dermatophagoides pteronyssinus and

Dermatophagoides farinae than chronic sinusitis patients There was no significant

difference of serum sIgE between chronic sinusitis patients and controls

2.5 Inflammatory Cell Scores in Nasal Polyps and Chronic Sinusitis

2.5.1 Inflammatory Cell Scores in Nasal Polyps and Its Paired Middle Turbinate, and Middle Turbinate of Allergic Rhinitis Patients and Controls

II Anti-CD8 (CD8+ T cell) staining of nasal polyps and its paired middle turbinate

Figure 9 (I to VII) Immunohistochemistry staining of CD4+ and CD8+ T cells, eosinophils, neutrophils, mast cells, CD19+ B cells and CD1a+ langerhans cells in nasal polyp (A) and its paired middle turbinate (B) Positive cells are stained with dark brown All the pictures are taken

under 100 × light microscope

Figure 9, Continued

A Nasal Polyps B The paired middle turbinate.

VI Anti-CD1a (langerhans cell) staining of nasal polyp and its paired middle turbinate

VII Anti-CD19 (B cell) staining of nasal polyp and its paired middle turbinate

Figure 9 (I to VII) shows immunohistochemical staining of CD4+ and CD8+ T cells,

CD19+ B cells, eosinophils, neutrophils, mast cells and CD1a+ langerhans cells in

nasal polyp tissue and the paired middle turbinate mucosa Figure 10 (I to VII) shows

the immunohistochemical staining in middle turbinate mucosa of allergic rhinitis

patients and controls These figures show that airway remodeling is commonly found

in nasal polyps and allergic rhinitis patients, including hyperplasia of epithelial cells,

basement membrane thickening and inflammatory cell infiltration All cell types were

mainly found in the lamina propria except langerhans cells which were mainly seen in

the epithelium Infiltration of eosinophils and CD8+ and CD4+ T cells could also be

found in the epithelium CD8+ and CD4+ T cells, eosinophils and mast cells were

distributed diffusely, especially when the level of cell infiltration was high

Neutrophils were numerous in the subepithelial region, and sometimes formed

clusters especially in patients with neutrophilia Langerhans cell and B cell numbers

were low in all the study groups

A Middle turbinate from allergic rhinitis B Middle turbinate from controls.

I Anti-CD4 (CD4+ T cell) staining of middle turbinate mucosa from allergic rhinitis

patients and controls

A B

II Anti-CD8 (CD8+ T cell) staining of middle turbinate mucosa from allergic rhinitis

patients and controls

Figure 10 (I to VII) Immunohistochemistry staining of CD4+ and CD8+ T cells, eosinophils,

neutrophils, mast cells, CD19+ B cells and CD1a+ langerhans cells in middle turbinate of

allergic rhinitis patients (A) and controls (B) Positive cells are stained with dark brown All

the pictures are taken under 100× light microscope

Figure 10, Continued

A Middle turbinate from allergic rhinitis B Middle turbinate from controls.

allergic rhinitis patients and controls

IV Anti-neutrophil elastase (neutrophil) staining of middle turbinate mucosa from

allergic rhinitis patients and controls

V Anti-tryptase (mast cell) staining of middle turbinate mucosa from allergic rhinitis

patients and controls

Figure 10, Continued

A Middle turbinate from allergic rhinitis B Middle turbinate from controls.

VI Anti-CD1a (langerhans cell) staining of middle turbinate mucosa from allergic

rhinitis patients and controls

VII Anti-CD19 (B cell) staining of middle turbinate mucosa from allergic rhinitis patients

and controls

2.5.1.2 Statistical Analysis

I Occurrence of high inflammatory cell scores in different study groups

Inflammatory cell infiltration in nasal polyps and its paired middle turbinate, and

middle turbinate from allergic rhinitis and controls was obtained by counting cells

under a light microscope of 400 times magnification Scores were recorded

accordingly The occurrence of high scores (score 2 or 3) of inflammatory cells in

different study groups is reported in Table 12

Table 12 Percentage of patients with high scores (score 2 or 3) of inflammatory cells

in nasal polyp tissue and paired middle turbinate (n=48), middle turbinate from allergic rhinitis (n=15) and control group (n=14)

Nasal polyps (n=48) Incidence rate of

high score

Polyp tissue MT (NP)1

MT (AR)2 (n=15)

MT (CON)3 (n=14)

a high score of the inflammatory cell studied and its percentage in the study group

Eosinophils, CD4+ and CD8+ T cells, and neutrophils were the main inflammatory

cells infiltrated in nasal polyp tissue and the paired middle turbinate 30 out of 48

(62.5%) nasal polyp patients had eosinophilia Meanwhile, 23 out of these patients

(76.7%) also had high eosinophil scores in the paired middle turbinate Eosinophilia

(score ≥2) was found in 63.6% (7/11) of patients with atopy and 73.1% (19/26) of patients without atopy Among the 30 nasal polyp patients who had a high score of

CD4+ T cell in the nasal polyp tissues, 23 (47.9%) patients also had a high score in

the paired middle turbinate There was also one nasal polyp patient (2.1%) who had a

high CD4+ T cell score in the middle turbinate, but a low score in the polyp tissue 23

(47.9%) nasal polyp patients had a high score of CD8+ T cell both in the nasal polyps

and the paired middle turbinate, but there were also 6 (12.5%) patients who had a high

score in the middle turbinate only 24 out of 48 (50%) nasal polyp patients had a high

neutrophil score in the polyp tissue Among these patients, 37.5% had neutrophilia in

the paired middle turbinate also In addition, 6 out of 48 (12.5%) nasal polyp patients

had neutrophilia in middle turbinate only These findings suggested that inflammatory

cell infiltration in nasal polyps and paired middle turbinate often coexisted in our

study patients

Mast cells had moderately infiltrated nasal polyp tissue and its paired middle turbinate

21 out of 48 (43.8%) patients had a high mast cell score in polyp tissue Among them,

15 (71.4%) also had a high score in the paired middle turbinate Another 6 (12.5%)

patients had a high score in middle turbinate but not in the polyp tissue CD19+ B cell

and CD1a+ langerhans cell infiltration was low in both nasal polyp and its paired

middle turbinate The incidence rate of a high CD19+ B cell score was 16.7% in polyp

patients 3 (6.25%) nasal polyp patients had a high score both in nasal polyp and the

paired middle turbinate Another 3 (6.25%) patients had a high CD19+ B cell score

only in the middle turbinate 7 patients (15.6%) had a high CD1a+ langerhans cell

score Four of them (57.1%) had a high score in the paired middle turbinate as well

In the middle turbinate of allergic rhinitis, CD8+ T cell was the major inflammatory

cell The incidence rate of patients with a high CD8+ T cell score was 60% Moderate

infiltration by CD4+ T cells, neutrophils and mast cells was seen, all with incidence

rates of 40% The incidence rate of eosinophilia was 26.7% CD19+ B cell and

langerhans cell were mildly infiltrated with incidence rates of 6.7% and 26.7%,

respectively The most commonly found inflammatory cell in the control group was

CD4+ T cells The incidence rate of controls with a high CD4+ T cell score was

42.9% CD8+ T cells and mast cells were moderately infiltrated in the control group

with incidence rates of 35.7% and 28.6%, respectively Other inflammatory cells in

controls, including eosinophils, neutrophils, CD19+ B cells as well as langerhans cells

were rarely found in the controls

II Correlation between inflammatory cells and serum IgE

Nonparametric Spearman correlation was used to analyze the correlation of

inflammatory cells to serum tIgE and sIgE None of the inflammatory cells in the

study groups had any correlations with serum tIgE or sIgE

III Correlation between inflammatory cells in the same sample

The correlation between different inflammatory cell scores was analyzed by

nonparametric Spearman correlation test The obtained significant correlations are

shown in Table 13 and Table 14

There was a strong, positive and significant correlation between CD4+ and CD8+ T

cell scores both in the nasal polyp and the middle turbinate of nasal polyp patients

Significant but moderate correlations were found between CD4+ T cells and

neutrophils, between CD19+ B cells and CD1a+ langerhans cells, and between

eosinophils and langerhans cells in nasal polyp tissue and the middle turbinate of

nasal polyp patients Besides these correlations, there were more correlations found in

the middle turbinate of nasal polyp patients than in the polyp tissues The obtained

correlations included correlations between CD4+/CD8+ T cells and B cells, between

CD4+ T cells/CD8+ T cells/B cells and mast cells, between langerhans cells and mast

cells, and between eosinophils and neutrophils

Table 13 Significant nonparametric Spearman correlations between different

inflammatory cell scores in the same sample (nasal polyp tissue and middle turbinate

of nasal polyp patients)

Subjects Nonparametric

Spearman correlation Sig (2-tailed)

Nonparametric Spearman correlation coefficient

The allergic rhinitis group was the only one that did not show any significant

correlation between CD4+ and CD8+ T cells Instead, a correlation between CD4+ T

cells and eosinophils was identified In the controls, besides the correlation between

CD4+ and CD8+ T cells, langerhans cells and eosinophils, a correlation between

neutrophils and mast cells was evidenced

Table 14 Significant nonparametric Spearman correlations between different inflammatory

cell scores in the same sample (middle turbinate from allergic rhinitis and controls)

Subjects Nonparametric

Spearman correlation Sig (2-tailed)

Nonparametric Spearman correlation coefficient

IV Median and Mean±SD

Table 15 Median and 95% confidence interval (mean±SD) of cell scores in nasal polyp

patients (n=48), allergic rhinitis (n=15) and controls (n=14)

Nasal Polyp

(n=48)

Allergic Rhinitis (n=15)

Controls

(n=14)

Cell studied

Polyp tissue Median (Mean±SD)

Paired middle turbinate Median (Mean±SD)

Middle turbinate Median

(Mean±SD)

Middle turbinate Median (Mean±SD)

CD4+ T cell

2 (1.7±1.2)

1.5 (1.5±1.1)

1 (1.2±1.3)

1 (1.4±1.3) CD8+ T cell

2 (1.9±1.2)

2 (1.9±1.1)

3 (1.9±1.4)

1 (1.2±1.2) CD19+ B cell

0.5 (0.7±0.9)

0 (0.6±0.9)

0 (0.3±0.8)

0 (0) Langerhans cell

0.5 (0.8±1.0)

0 (0.6±0.8)

0 (0.7±1.2)

0 (0.07±0) Eosinophil

2 (1.9±1.2)

1 (1.5±1.1)

0 (0.7±1.0)

0 (0.4±0.6) Neutrophil

1.5 (1.5±1.1)

1.5 (1.7±1.1)

1 (1.3±1.4)

0 (0.4±0.6) Mast cell

1 (1.4±1.0)

1 (1.5±1.0)

1 (1.1±0.9)

1 (1.3±0.7)

Table 15 shows the median score and the 95% confidence interval of cell scores in

nasal polyp patients, allergic rhinitis and controls In the nasal polyp tissue and the

paired middle turbinate mucosa, the predominant cell infiltrations were CD8+ and

CD4+ T cells, eosinophils and neutrophils In the allergic rhinitis patients, the

infiltration of CD8+ T cells was prominent, followed by CD4+ T cells, neutrophils,

mast cells and eosinophils In the control group, the main residential cell types were

CD4+ and CD8+ T cells, followed by mast cells CD19+ B cells and langerhans cells

were rarely seen in any study group

V Inflammatory score analysis of nasal polyp and its paired middle turbinate

Table 16 (I to VII) shows cell score distribution in the paired samples from nasal

polyp patients (nasal polyp tissue and the middle turbinate from the same side)

Statistic analysis indicated that inflammatory cell distribution was well correlated

between nasal polyp and its paired middle turbinate (Table 17)

NP (MT) 1

NP 2

I CD4+ T cells II CD8+ T cells

Table 16 (I to VII) Cell score distribution in the paired samples from nasal polyp patients (nasal

polyp tissue and middle turbinate from the same side, n=48) NP (MT)1, inflammatory cell score in middle turbinate from nasal polyp patients NP, inflammatory cell score in nasal polyp tissue Numbers in the cells indicate patient number

Table 17 Distribution and correlation of the inflammatory cell scores in nasal polyp and its paired

Asym

Sig

(2-tailed)

Nonparametric Spearman’s correlation P value

Correlation Coefficient

Table 17 shows the result of inflammatory cell distribution and correlation analysis

between nasal polyp and its paired middle turbinate The results indicated that

inflammatory cell distribution was well correlated between nasal polyp and its paired

middle turbinate All the inflammatory cells studied, i.e., eosinophils, CD4+ and

CD8+ T cells, neutrophils, mast cells, CD19+ B cells and langerhans cells were

significantly, positively and strongly correlated between nasal polyp and its paired

middle turbinate In addition, the cell distributions were similar in the paired samples

There was no significant difference in cell distribution between the two subjects

except for a significantly higher eosinophil score in the nasal polyp tissue than in the

paired middle turbinate

VI Inflammatory cell score in different study groups

Table 18 shows that nasal polyp tissue and the paired middle turbinate had

significantly higher scores of eosinophils, CD8+ T cells, neutrophils, CD19+ B cells

and langerhans cells than the middle turbinate from controls Nasal polyp tissue also

had significantly higher scores of CD8+ T cells and CD19+ B cells than the middle

turbinate of allergic rhinitis patients There was no significant difference in CD4+ T

cell and mast cell scores between nasal polyp patients and allergic rhinitis

patients/controls

Table 18 Wilcoxon rank sum test for 2-independent samples between nasal polyp patients (n=48),

allergic rhinitis (n=15) and controls (n=14)

Nasal polyp tissue Middle turbinate(NP)1Subjects

Wilcoxon rank sum test Z value

Asym Sig

(2-tailed)

Wilcoxon rank sum test Z value

Asym Sig (2-tailed) Middle turbinate (AR)2 -1.238 0.216 -0.801 0.423 CD4+ T cell

Middle turbinate (CON)3

Middle turbinate (AR) -0.149 0.882 -0.344 0.731 CD8+ T cell

Middle turbinate (CON)

-2.005 0.045 -1.985 0.047

Middle turbinate (AR) -2.297 0.022 -1.736 0.083 CD19+ B cell

Middle turbinate (CON)

-2.845 0.004 -2.401 0.016

Middle turbinate (AR) -3.192 0.001 -2.383 0.017

Eosinophil

Middle turbinate (CON)

-4.018 <0.0001 -3.533 <0.0001

Middle turbinate (AR) -0.726 0.468 -1.233 0.218 Neutrophil

Middle turbinate (CON)

-3.462 0.001 -3.95 <0.0001

Middle turbinate (AR) -0.856 0.392 -1.184 0.236 Mast cell

Middle turbinate (CON)

-1.76 0.86 -0.516 0.606 Middle turbinate (NP) 1 , middle turbinate from nasal polyp patients; Middle turbinate (AR) 2 , middle turbinate from allergic rhinitis patients; Middle turbinate (CON) 3 , middle turbinate from controls

VII Scatter figures

Figure 11 Scatter figures (with mean) of the scores of CD4+ T cells, CD8+ T cells, eosinophils,

neutrophils, mast cells, CD19+ B cells and langerhans cells in nasal polyps and the paired middle turbinate (n=48), middle turbinate from allergic rhinitis patients (n=15) and controls (n=14) P value with significance (<0.05) is indicated MT(NP)*: Middle turbinate from nasal polyp patients MT(AR) * :Middle turbinate from allergic rhinitis MT (CO)*: Middle turbinate from controls P1, P value of nonparametric Spearman correlation test (2-tailed) P2, P value of Wilcoxon signed rank test; P3: P value of Wilcoxon rank sum test r: Correlation coefficient of nonparametric Spearman correlation

P1<0.0001, r=0.625

P3<0.01 P3<0.01

VIII Pattern of combined inflammatory cell infiltration in nasal polyp

Nasal polyps are mainly classified into eosinophilic and neutrophilic nasal polyps

Eosinophils, neutrophils and mast cells are reported to be the main inflammatory cells

in the pathogenesis of nasal polyps However, an indication of the pattern of

combined inflammatory cell expression has been lacking In our study, there was no

clear pattern in this series, and combinations of cell types were more common than

single cell types In addition, lymphocytes, especially CD8+ T cells, highly infiltrated

in both nasal polyp tissue and the paired middle turbinate Figures 12 and 13 show

the pattern of infiltration of these cells with a cell score equal to or greater than two

Inflammatory Cell Pattern in Nasal Polyps (Eosinophil, Neutrophil and Mast Cell)

Eos

13%

Neu10%

Figure 12 Pattern of eosinophil, neutrophil and mast cell infiltration in all nasal polyp

patients (n=48, 100%) This figure shows the pattern of infiltration of these cells with cell score equal to or greater than two Eos, eosinophil; Neu, neutrophil; MC, mast cell

Figure 12 shows the cell pattern (scores ≥2) of eosinophils, neutrophils and mast cells

in nasal polyp tissues Eosinophils were the predominant cells, being present in 63%

(n=30) of all the nasal polyp patients The largest single group consisted of

eosinophils alone, in 10 patients (21%) In addition, eosinophils were combined with

neutrophils in eight patients (17%); and with mast cells in four patients (8%); with

both neutrophils and mast cells in a further eight patients (17%) Mast cells alone and

neutrophils alone were seen in six and five patients, respectively (13% and 10%)

Mast cells and neutrophils were combined in three patients (6%) In four patients

(8%), none of these cells was seen

Inflammatory Cell Pattern in Nasal Polyp (CD8+ T Cell, Eosinophil and Neutrophil)

None

CD8 6%

Neu 11%

CD8+Eos Eos+Neu

23%

17%

Eos

CD8+Neu 6%

6%

CD8+Eos+Neu 17%

14%

Figure 13 Pattern of CD8+ T cell, eosinophil and neutrophil infiltration in all nasal polyp

patients (n=48, 100%) This figure shows the percentage of each of these cells with cell score

equal to or greater than two Eos, eosinophil; Neu, neutrophil; CD8, CD8+ T cell

According to our data analysis, CD8+ T cells, eosinophils and neutrophils were the

major inflammatory cells in nasal polyps Figure 13 shows the cell pattern (scores ≥2)

of these three cells in nasal polyp tissue 29 (60%) of the patients had high score of

CD8+ T cells Among them, 7 (14%) of the patients had CD8+ T cells alone; 11 (23%)

of the patients had combined infiltration of CD8+ T cells and eosinophils; 3 (6%) of

the patients had a combination of CD8+ T cells and neutrophils; 8 (17%) of the

patients had CD8+ T cells, eosinophils and neutrophils all together 3 (6%) and 5

(11%) of the patients had respectively eosinophils or neutrophils alone Only 3

patients (6%) did not have any of these cells with a score equal to or above two

2.5.2 Inflammatory Cell Scores in Inflamed Sinus Mucosa and its Paired

Middle Turbinate, Middle Turbinate from Allergic Rhinitis Patients and Controls

2.5.2.1 Immunohistochemistry Staining

Figure 14 (I to VII)shows immunohistochemical staining of CD4+ and CD8+ T cells, CD19+ B cells, eosinophils, neutrophils, mast cells and CD1a+ langerhans cells in

inflamed sinus mucosa and the paired middle turbinate The character of

inflammatory cell distribution was similar to that of nasal polyps

A Inflamed sinus mucosa B Paired middle turbinate.

I Anti-CD4 (CD4+ T cell) staining of inflamed sinus mucosa and its paired middle turbinate

II Anti-CD8 (CD8+ T cell) staining of inflamed sinus mucosa and its paired middle turbinate

Figure 14 (I to VII) Immunohistochemistry staining of CD4+ and CD8+ T cells, eosinophils, neutrophils, mast cells, CD19+ B cells and langerhans cells in inflamed sinus mucosa (A) and the paired middle turbinate (B) Positive cells are stained with dark brown All the pictures are taken

under 100× light microscope

Figure 14, Continued

A Inflamed sinus mucosa B Paired middle turbinate.

and its paired middle turbinate