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

Chapter 2. Inflammatory Cell Patterns in
Nasal Polyps and Chronic Sinusitis

2.1 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
135
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
136
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.
137
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
environmental factors.


2.3 Methodology
2.3.1．Working Definitions
Guidelines in the diagnosis of chronic sinusitis, nasal polyps, allergic rhinitis and
atopy are:
I. Chronic sinusitis
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
138
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
139
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
140
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
Nasal polyps 44 48 34/14
Chronic sinusitis 47 20 10/10
Allergic rhinitis 27 15 14/1

Control patients 27 14 11/3

2.3.3 Immunohistochemical study
2.3.3.1 Collecting Samples
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
141
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% H
2
O
2
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
142
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.
Antibody Manufacturer Clone Specificity
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)
Anti-CD1a DakoCytomation NA1/34 Langerhans cell
Tryptase DakoCytomation AA1 Mast cell
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;
143
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.


2.3.4 Allergy Test
Table 10. Specific IgE interpretation.
29


Quantitative Results
( kU/L)

Semi-Quantitative Results
(Specific IgE Class)

Level of Allergen Specific
Antibody
<0.35 0 Absent/Undetectable
0.35-0.7 1 Low
0.7-3.5 2 Moderate
3.5-17.5 3 High
17.5-50 4 Very high
50-100 5 Very high
>100 6 Very high

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.

144
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
145
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.
146
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

A. Anti-CD3 B. Anti-CD4

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

147
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.

2.4.2 Histopathology Changes

A. B.

C. D.
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
148
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.

149
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).
Subject Atopy*
tIgE≥100
tIgE<100
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
150
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
151
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
2.5.1.1 Immunohistochemistry Staining

A. Nasal Polyps. B. The paired middle turbinate.


I. Anti-CD4 (CD4+ T cell) staining of nasal polyps and its paired middle turbinate.

A. B.


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.
152
Figure 9, Continued.

A. Nasal Polyps. B. The paired middle turbinate.


III. Anti-eosinophil major basic protein (eosinophil) staining of nasal polyp and its paired
middle turbinate.

A. B.


IV. Anti-neutrophil elastase (neutrophil) staining of nasal polyp and its paired middle
turbinate.

A. B.

V. Anti-tryptase (mast cell) staining of nasal polyp and its paired middle turbinate.

153
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.

A. B.

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
154
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.
155
Figure 10, Continued.

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


III. Anti-major basic protein (eosinophil) staining of middle turbinate mucosa from
allergic rhinitis patients and controls.

A. B.

IV. Anti-neutrophil elastase (neutrophil) staining of middle turbinate mucosa from
allergic rhinitis patients and controls.

A. B.

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

156
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.

A. B.

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.
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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)
Eosinophil 30 (62.5%)
4
23 (47.9%) 4 (26.7%) 1 (7.1%)
CD8+ T cell 29 (60.4%) 28 (58.3) 9 (60%) 5 (35.7%)
CD4+ T cell 30 (62.5%) 24 (50%) 6 (40%) 6 (42.9%)
Neutrophil 24 (50%) 24 (50%) 6 (40%) 1 (7.1%)
Mast cell 21 (43.8%) 21 (43.8%) 6 (40%) 4 (28.6%)
Langerhans cell

7 (15.6%) 6 (12.5%) 4 (26.7%) 0
CD19+ B cell 8 (16.7%) 6 (12.5%) 1 (6.7%) 0
MT (NP)
1
, middle turbinate of nasal polyp patients. MT (AR)
2
, middle turbinate of allergic rhinitis
patients. MT (CON)
3
, middle turbinate of control group. 4, entries are the number of patients with
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
158
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%,
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