Keywords
paranasal sinus disease - nasal mucosa - CD4-positive T-lymphocytes
Introduction
Chronic rhinosinusitis (CRS) is a frequently encountered disease in daily otorhinolaryngology
practice. Patients with CRS present with nasal discharge, postnasal drip, and nasal
congestion.[1] CRS is commonly classified based on the presence or absence of nasal polyps (NPs).[2] Patients without NPs (CRSsNP) are known to respond to treatment better than CRS
patients with NPs (CRSwNP). Additionally, the prognosis of most CRSwNP patients is
poor and refractory.[2] As a result, the quality of life of patients with CRSwNP is extremely low due to
chronic olfaction disorder and severe nasal congestion.[3]
[4]
In most cases of CRSwNP in Europe and in the United States, eosinophilic infiltration
in the inflammatory regions of the nose and paranasal sinuses is observed.[2]
[5]
[6] However, infiltration of neutrophils and lymphocytes without eosinophils is commonly
observed in the NPs of CRSwNP patients in East Asian countries, such as Korea and
Japan.[7]
[8] Since eosinophils, in combination with T-helper 2 (Th2) cells, play a role in allergic
immune response,[9] the presence of eosinophilic infiltration, but not NPs, appears to be one of the
exacerbation factors of CRS. Therefore, distinction of CRSwNP based on the presence
or absence of eosinophils in the NPs has been clinically considered important, especially
in East Asian countries.
Accordingly, a disease category, eosinophilic CRS (ECRS) has been introduced to represent
CRSwNP patients with eosinophilic infiltration in the NPs. Clinically, ECRS and non-ECRS
(NECRS) can be classified based on the guidelines of the Japanese Epidemiological
Survey of Refractory Eosinophilic Chronic Rhinosinusitis (JESREC), according to which
assessment of computed tomography findings, eosinophil ratio in the peripheral blood,
and the presence or absence of NPs can be addressed.[10] However, information on the infiltrated immune cells, other than eosinophils, in
the NPs of ECRS patients is scant, maybe because the main focus has always been on
the presence of eosinophils in the NPs.[11]
The epithelia of the nasal and paranasal sinuses not only are the important first
barriers in the body, but also respond to external stimuli and induce diverse immune
responses.[12] However, the immune status, especially the involvement of acquired immunity in the
paranasal sinus mucosa in ECRS patients, has not been well documented. To clarify
the different types of lymphocytes infiltrated in NPs between ECRS and NECRS, we isolated
the infiltrating cells from the surgically removed polyps in the paranasal sinus mucosa
of CRSwNP patients and found that the CD4+ T-cell/B-cell ratio was significantly higher in ECRS patients than in NECRS patients.
Therefore, the CD4+ T-cell/B-cell ratio could be an informative indicator for ECRS.
Materials and Methods
Patients
All patients included in the study signed informed consent forms. The study was approved
by the ethics committees of Toho University School of Medicine (27028 and A18086_27028).
The present study included 18 patients with CRSwNP who were operated at Toho University
Omori Medical Center. We divided CRSwNP patients into two subgroups, ECRS (≥ 11 points)
and NECRS (≤ 10 points) based on the JESREC scoring system[10] before surgery. We histologically confirmed the presence of eosinophils in the NPs
of the ECRS patients.
Preparation of Nasal Sinus Mononuclear Cells
The uncinate processes of anterior ethmoid sinus of patients who underwent endoscopic
sinus surgery were obtained. The mucosa specimens from each patient were minced and
incubated with Hanks' balanced salt solution (HBSS) containing 5% fetal calf serum,
0.5 mg/ml collagenase A (Roche, Basel, Switzerland), 0.2 mg/ml DNase I (Sigma-Aldrich,
St. Louis, MO, USA), and 10 mM hydroxyethyl piperazine ethanesulfonic acid (HEPES)
at 37°C for 30 minutes. After incubation, 100 mM ethylenediaminetetraacetic acid (EDTA)
was added to stop the enzyme reaction, and the specimens were passed through a 70-µm
filter (BD Bioscience, Franklin Lakes, NJ, USA). After centrifugation, the cells were
resuspended in 10 ml of 40% (v/v) Percoll (GE Healthcare Bioscience, Uppsala, Sweden)
and overlaid with 2 ml of 80% (v/v) Percoll. Subsequently, the suspension was continuously
centrifuged at 2,400 rpm for 20 minutes at 25°C. Mononuclear cells were collected
from the interface.
Flow Cytometry
The following antibodies were used for cell-surface staining: anti-CD4 (OKT4) antibodies
labeled with fluorescein isothiocyanate (FITC), and CD8 (RPA-T8) antibodies conjugated
with APC-Cy7 from TONBO Bioscience (San Diego, CA, USA). Anti-CD20 (2H7) antibodies
with PE-Cy7 were purchased from Bio Legend (San Diego, CA, USA). Cells prepared as
described above were stained with the mixture of labeled antibodies on ice for 20 minutes.
After washing, cells were fixed with paraformaldehyde and analyzed with FACS Canto
II flow cytometer (BD Bioscience). The data were analyzed with Flow Jo software (Tree
Star, Ashland, OR, USA).
Statistical Analysis
Statistical analysis was performed using the IBM SPSS Statistics Version 25.0. software
(IBM Corp., Armonk, NY, USA). Comparison between 2 groups was performed using the
Mann-Whitney U test. P-values < 0.05 were considered to be statistically significant.
Results
ECRS is a subtype of CRSwNP with eosinophil infiltration in the NPs. But it had not
been clearly clarified types of lymphocytes infiltrated in NPs of ECRS patients. To
address this issue, we isolated mononuclear cells in NPs from NECRS and ECRS patients
and analyzed cell types by a flow cytometry. In this study, a total of 18 patients
with CRSwNP (12 male, 6 female), with a mean age of 56.61 ± 6.79 years old and a mean
blood eosinophil count of 8.49 ± 5.82%, were included. Of the 18 patients, 6 had NECRS
and 12 had ECRS.
First, the lymphocytes infiltrating the paranasal sinus mucosa were analyzed in NECRS
and ECRS groups. Mononuclear cells were isolated from the paranasal sinus mucosa,
were stained with anti-CD4, anti-CD8, and anti-CD20 antibodies, and were analyzed
on FACS. As shown in [Fig. 1], CD8+ T-cells were dominant over CD4+ T-cells in both NECRS and ECRS. This is different from the secondary lymphoid organs,
in which more CD4+ T-cells are present than CD8+ T-cells.[13] Based on the percentage of each cell population, it is possible to calculate absolute
cell numbers and standardize the numbers according to the weight and/or size of the
collected tissues. But it must be very difficult because recovery efficiency of the
infiltrating cells from the tissue might be different in each preparation and/or sample.
Therefore, we utilized the percentage of cell populations in the FACS analysis and
looked for the combination of populations whose ratio between NECRS and ECRS would
be significant.
Fig. 1 Representative fluorescence-activated cell sorter (FACS) plots of infiltrating cells
in the paranasal sinus mucosa. Infiltrating cells in the paranasal sinus mucosa of
NECRS (a) and ECRS (b) patients were collected and were analyzed using FACS. The number shown in figures
is the percentage of cells in each gate indicated.
As shown in [Fig. 2], no significant differences were observed in the CD8+ T-cell/B-cell or CD4+ T-cell/CD8+ T-cell ratios between ECRS and NECRS patients; however, the CD4+ T-cell/B-cell ratio was significantly higher in ECRS patients than in NECRS patients
(mean value = NECRS 0.95 vs ECRS 4.15, Mann-Whitney U test for independent samples,
p = 0.032) ([Fig. 2b]). Thus, the relative increase in the counts of CD4+ T-cells infiltrating the paranasal sinus mucosa in ECRS patients reflected the diagnostic
criteria for ECRS, which is associated with a high recurrence rate and a high proportion
of refractory cases.
Fig. 2 The CD4+ T-cell/B-cell ratio was significantly different between ECRS and NECRS patients.
CD4+ T-cell/CD8+ T-cell ratio (a), CD4+ T-cell/B-cell ratio (b), and CD8+ T-cell/B-cell ratio (c) in the paranasal sinus mucosa of NECRS and ECRS patients were determined. Each ratio
was compared between NECRS and ECRS (*: p < 0.05). All data expressed as median interquartile range.
Discussion
Moderate and severe ECRS, defined based on the JESREC scoring system, are commonly
concomitant with bronchial asthma; however, the pathogenic mechanism of ECRS remains
unclear.[10] Therefore, it is important to understand the clinical features of ECRS to establish
a better treatment strategy for ECRS. Since CRSwNP in Europe and the United States
is generally associated with poor prognosis,[2] the pathogenesis of CRSwNP has been extensively investigated. Immunostaining of
NPs in patients with CRSwNP showed infiltration of eosinophils, CD4+ T-cells, and CD68+ macrophages, and these cells produced interleukin 17A, which might be one of the
factors that causes inflammation.[14]
[15] Additionally, compared with normal inferior turbinate mucosa, NPs in patients with
CRSwNP showed infiltration of significantly higher counts of CD4+ T-cells.[16] These results suggested that CD4+ T-cells in NPs were strongly involved in the pathogenesis of CRSwNP. In contrast,
we and others[17]
[18] demonstrated that the CD8+ T-cell count was higher than the CD4+ T-cell count in the paranasal sinus mucosa of patients with CRSwNP, compared with
that of patients with CRSsNP ([Fig. 1]). Although it has been suggested that CD8+ T-cells that infiltrated the paranasal sinus mucosa in patients with CRSwNP are largely
effector-memory phenotype cells,[17]
[19] the reason for high counts of CD8+ T-cells in the paranasal sinus mucosa of patients with CRSwNP and the functions of
these cells remain unclear. This CD8+ T-cell dominance was observed in NPs irrespective of the presence of eosinophilic
infiltration ([Fig. 1]).
In contrast, the CD4+ T-cell/B-cell ratio in the infiltrated cells in the paranasal sinus mucosa of CRSwNP
patients was significantly higher in ECRS than in NECRS, indicating that the proportion
of cell population infiltrating the paranasal sinus mucosa was highly correlated with
the CRS classification developed by JESREC. In the past, the activation of CD4+ T-cells and dysfunction of the regulatory T-cells have been shown to play important
roles in the pathogenesis of CRS.[20]
[21] Particularly, local CD4+ T-cells have been found to exhibit different phenotypes of CRS by producing different
types of cytokines.[16] ECRS has a higher rate of recurrence (more than double) and intractable percentage
(more than triple) than NECRS.[10] Thus, CD4+ T-cells might be more involved in the pathogenesis of ECRS than in that of NECRS.
In the present study, we could not investigate the functionality of CD4+ T-cells due to the limitation of samples. Further studies must be conducted to obtain
insights into the pathogenesis of ECRS.
Conclusion
In the present study, we found that the CD4+ T-cell/B-cell ratio in the paranasal sinus mucosa was significantly higher in ECRS
patients than in NECRS patients. Hence, the CD4+ T-cell/B-cell ratio can be used as a potential indicator for differentiating between
ECRS and NECRS. Further studies are necessary to clarify the role of mucosal CD4+ T-cells in the pathogenesis of ECRS.
