cytokine profiles in japanese patients

Allergology International Vol 61, No1, 2012 www.jsaweb.jp 115

Cytokine Profiles in Japanese Patientswith Chronic RhinosinusitisTakayuki Sejima1,2, Gabriele Holtappels1, Hisashi Kikuchi2, Shoichiro Imayoshi2,Keiichi Ichimura2 and Claus Bachert1

ABSTRACTBackground: Chronic rhinosinusitis (CRS) is classified in CRS without nasal polyp (CRSsNP) and CRS withnasal polyp (CRSwNP) in western countries, whereas this classification was not common so far in Japan.Studying inflammatory mediators in clearly defined disease subgroups may lead to a better differentiation ofchronic sinus diseases.Methods: Homogenates of sinonasal mucosal tissue from 14 controls, 9 CRSsNP patients, and 19 CRSwNPpatients were assayed for transforming growth factor (TGF)-, interleukin (IL)-5, immunoglobulin E (IgE),Staphylococcus enterotoxin (SAE)-IgE, eosinophil-catioic protein (ECP), myeloperoxidase (MPO), IL-1, IL-6,and IL-8 by enzyme-linked immunosorbent assay or UNICAP system.Results: CRSwNP had significantly higher levels of IL-5, IgE, SAE-IgE, and ECP compared with CRSsNP andcontrols. CRSsNP was characterized by high levels of TGF-, while CRSwNP showed a Th2 polarization andlower levels of TGF-. Especially, in CRSwNP samples, 68.4% were eosinophilic (ECPMPO ratio >1), and52.6% were SAE-IgE positive. On the other hand, in 9 CRSsNP patients, eosinophilic or SAE-IgE positive sam-ple was only one sample respectively. Additionally, 31.6% of CRSwNP were asthmatic patients, while none ofCRSsNP patient was suffering from bronchial asthma.Conclusions: This study is thought to be the first one that showed the cytokine profiles in Japanese CRSswNP similar to those of European CRS. Based on mediator profiles, we suggest that CRSsNP and CRSwNPare distinct disease entities within the group of chronic sinus diseases.

KEY WORDSchronic sinusitis, ECP, inflammation, nasal polyps, TGF-

INTRODUCTIONChronic rhinosinusitis (CRS) is one of the most com-mon diseases in the otolaryngology area. In westerncountries including Europe and America, the nasalpolyp is considered to be the result of eosinophilic in-flammation in nasal mucosa, and it is generally ac-cepted that CRS is classified in CRS without nasalpolyp (CRSsNP) and CRS with nasal polyp(CRSwNP). Recent research has demonstrated thatthe pathologies of CRSsNP and CRSwNP can be dif-ferentiated into distinct subgroups on the basis of theexpression of inflammatory mediators1 and histopa-thology.2,3 On the other hand, in former Japan, CRScaused by bacterial infection held an overwhelming

majority, and the main inflammatory cell which infil-trated in tissues including nasal polyps was the neu-trophil.4 The CRS classification such as CRSsNP orCRSwNP was not common so far in Japan. At present,however, characteristics of nasal polyp in Japan re-semble those in Europe and America, and eosino-philic infiltration is a more common feature histologi-cally.4 Such classification based on the pathophysiol-ogy of the disease may be useful for the decision forthe treatment.CRS is characterized by chronic inflammation of

the nasal and paranasal mucosa, accompanied by tis-sue remodeling that includes changes in the extracel-lular matrix (ECM) protein deposition and tissuestructure.2 In western countries, CRSsNP is charac-

Allergology International. 2012;61:115-122

ORIGINAL ARTICLE

1Upper Airway Research Laboratory, Department of Otorhino-laryngology, Ghent University Hospital, Ghent, Belgium and 2De-partment of Otorhinolaryngology-Head & Neck Surgery, JichiMedical University, Tochigi, Japan.Conflict of interest: No potential conflict of interest was disclosed.Correspondence: Takayuki Sejima, MD, PhD, Department of Otor-

hinolaryngology, Jichi Medical University, Yakushiji 33111 Shi-motsuke, Tochigi 3290498, Japan.Email: [email protected] 8 November 2010. Accepted for publication 17 July2011.2012 Japanese Society of Allergology

DOI: 10.2332allergolint.10-OA-0290

Sejima T et al.

116 Allergology International Vol 61, No1, 2012 www.jsaweb.jp

Table1Patient characteristics and symptom scores

Controls subjects

CRSsNP patients

CRSwNP patients

ANOVA (Fisher)

No. 14 9 19Age (years; range) 62.5 (21-80) 57 (29-71) 56 (17-71)Female/male sex 4/10 4/5 4/15 0.216Atopic status 0/14 2/9 7/19 0.011Asthma 0/14 0/9 6/19 0.020CT score 0 (0-1) 5 (2-10) 16 (10-20)

Cytokines in Chronic Rhinosinusitis


Fig.1Pathological fi ndings in mucosa. The sections were stained with hematoxylin-eosin in controls (A and B), CRSsNP (C and D), and CRSwNP (E and F), respectively. Arrows indicate eosinophils. Asterisks indicate pseudocyst formation. Bars; 20 m.

Control CRSsNP CRSwNP

HISTOPATHOLOGIC ANALYSISThe tissue was fixed with 10% neutral buffered forma-lin solution for 1 week, embedded in paraffin, and cutinto 2-m-thick sections. After hematoxylin-eosin(HE) staining, histopathologic analysis was per-formed by selecting a representative field of mucosawhere the most prominent change was detected with200 magnification. The number of eosinophils wasanalyzed by using a magnification 200. 5 fields of themucosal area were evaluated at random, and thenumber of eosinophilsfield was counted for eachsample by two independent evaluators.

MEASUREMENT OF CYTOKINE AND IgE LEV-ELSFreshly obtained tissue specimens were homoge-nized in Japan as previously described.12 For homog-enization, the tissue was thawed, weighed and 1 mLsaline was added per every 0.1 g tissue. The tissuewas then homogenized at 1000 rpm for 3 min on ice(homogeniser; B.Braun, Melsungen, Germany). Thehomogenates were centrifuged at 3000 rpm for 10min at 4. After centrifugation, supernatants werealiquot in 500 L and stored at -80 until analysis. Allsamples were assayed for IL-1, IL-5, IL-6, IL-8, IL-10,IL-17, IFN-, TGF- and MPO by using commerciallyavailable ELISA kits (Quantikine ELISA, R&D Sys-

tems, Minneapolis, MN, USA; MPO, Oxis Interna-tional, Portland, OR, USA). ECP, IgE and SAE-IgE(IgE to Staphylococcus enterotoxin A, C, and TSST-1; 0.35-100 kUAL) were measured by using theUNICAP system (Pharmacia, Uppsala, Sweden).These assays were performed in Belgium followingthe manufacturers instructions. An ECPMPO ratiowas calculated as described before.7 Standard curvesand controls were performed according to the guide-lines of the producer.

STATISTICAL ANALYSISStatistical analysis was performed with SPSS Statis-tics software v 17.0 (SPSS, Chicago, IL, USA). Dataare expressed as median, range and interquartiles.Statistical analysis was performed by using theKruskal-Wallis test and the Mann-Whitney U 2-tailedtest for unpaired comparisons. When comparisonswere made between groups, the Kruskal-Wallis testwas used to establish the significant intergroup vari-ability. The Mann-Whitney U test with with Bonfer-roni correction was then used for between-groupcomparisons. Baseline variables were analyzed by us-ing a 1-way ANOVA test or the Fisher exact test. Astatistical difference of P < 0.05 was considered sig-nificant.

Sejima T et al.

118 Allergology International Vol 61, No1, 2012 www.jsaweb.jp

Fig.2Measurement of transforming growth factor (TGF)-, interleukin (IL)-5, immunoglobulin E (IgE), Staphylococcus enterotoxin (SAE)-IgE, eosinophil-related mediators (ECP), myeloperoxidase (MPO), IL-1, IL-6, and IL-8 by enzyme-linked immunosorbent assay in 14 controls (CTR), 9 chronic rhinosinusitis without nasal polyp patients (CRSsNP), and 19 chronic rhinosinusitis with nasal polyp patients (CRSwNP). Values are reported as medians and interquartile ranges for each group. Statisti-cal analysis was performed using the Kruskal-Wallis test followed by a Mann-Whitney U-test with Bonferroni correction. The signifi cance level was set at a P value of 0.05. From these data, an ECP/MPO ratio was calculated. *P < 0.05; **P < 0.01.

P = 0.036*

P = 0.006*

CTR CRSsNP CRSwNP CTR CRSsNP CRSwNP





3500030000250002000015000100005000

0

17500150001250010000750050002500

0

262414126420

14000130001200011000300020001000

0

30000

20000

10000

0

400

300

200

100

0

1000

800

600

400

200

0

8

6

4

2

0

350300250200150100500

P = 0.036*

P = 0.018*

P = 0.036*

P = 0.018*

P = 0.003**P = 0.015*P = 0.024*

P = 0.031*

P = 0.021*

P = 0.028*

P = 0.009**

TGF- in tissue homogenatesP = 0.006**

IgE in tissue homogenatesP = 0.006**

SAE-IgE in tissue homogenatesP = 0.018*

ECP in tissue homogenatesP < 0.001**

IL-6 in tissue homogenates IL-8 in tissue homogenates

MPO in tissue homogenatesP = 0.026*

ECP/MPO ratioP = 0.027*

IL-1 in tissue homogenates

IL-5 in tissue homogenatesP = 0.018*

TG

F-

(pg/

mL)

IgE

(KU

/L)

EC

P (

g/L)

EC

P/M

PO

ratio

4000

3000

2000

1000

0

IL-6

(pg/

mL)

IL-1 (

pg/m

L)IL

-8 (p

g/m

L)S

AE

-IgE

(KU

/L)

MP

O (n

g/m

L)IL

-5 (p

g/m

L)

Cytokines in Chronic Rhinosinusitis


Table2Cytokine concentrations in CRSwNP subgroups with and without atopic status

CRSwNP with atopic status (n = 7) CRSwNP without atopic status (n = 12) P value

ECP (g/L) 27600 (3660-58500) 5750 (2960-13100) 0.033*MPO (ng/mL) 8875 (273-24683) 3703 (637-11558) 0.914IL-5 (pg/mL) 511 (54-720) 26 (6-37) 0.009**IFN- (pg/mL) 78 (78-78) 156 (78-1016) 0.298IgE (KU/L) 1070 (540-1265) 354 (55-477) 0.009**SAE-IgE (KU/L) 8.2 (5.2-14.3) 0 (0-3.8) 0.004**IL-1 (pg/mL) 37 (23-1097) 34 (27-410) 0.762IL-6 (pg/mL) 351 (303-1109) 95 (47-461) 0.258IL-8 (pg/mL) 5345 (2213-25243) 8693 (1550-25708) 0.904IL-17 (pg/mL) 11 (11-11) 11 (11-149) 0.285TGF- (pg/mL) 13837 (11635-18809) 13689 (9374-15239) 0.610

Data are expressed as medians and interquartile ranges. *P < 0.05 and **P < 0.01.

RESULTSPATIENT CHARACTERISTICClinical characteristics and symptom scores of all pa-tients are summarized in Table 1. A significant higherfrequency of asthma was reported in CRSwNP pa-tients, all other subjects were free of asthma symp-toms. One-way anova showed that the total symptomscore was the highest in CRSwNP patients of whichnasal congestion and loss of smell were the most pre-dominant symptoms. The scores for nasal congestionand loss of smell were significantly higher inCRSwNP vs CRSsNP patients, whereas CRSsNP pa-tients reported higher symptom scores for headachecompared with the other disease groups. Finally,CRSwNP showed the highest CT score comparedwith CRSsNP.

HISTOPATHOLOGIC ANALYSISHE-stained sections are shown in Figure 1. CRSwNPrevealed a thickened basement membrane, a subepi-thelial accumulation of eosinophils and edematouschange with pseudocyst formation (Fig. 1E, F). Onthe other hand, in CRSsNP, fibrosis and accumula-tion of not only eosinophils but also other inflamma-tory cells (neutrophils, lymphocytes and so on) weremostly found (Fig. 1C, D). The median (interquartilerange [IQR]) counts for eosinophils were signifi-cantly higher in CRSwNP (15; 12-17) compared toCRSsNP (6; 4-9; P = 0.02) and controls (1; 0-2; P

cytokine profiles in japanese patients

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