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Local IgE-mediated hypersensitivity to Alternaria in pediatric adenoid tissue
International Journal of Pediatric Otorhinolaryngology 76 (2012) 1423–1428
Contents lists available at SciVerse ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Local IgE-mediated hypersensitivity to Alternaria in pediatric adenoid tissue§ Seung Youp Shin a, Young Min Ye b, Young Gyu Eun a, Sung Wan Kim a, Joong Saeng Cho a, Hae Sim Park b,* a b
Department of Otorhinolaryngology-Head and Neck Surgery, Kyung Hee University School of Medicine, Seoul, Republic of Korea Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 October 2011 Received in revised form 13 June 2012 Accepted 14 June 2012 Available online 6 July 2012
Objective: Fungus may contribute to the development and exacerbation of allergic airway diseases. Several studies have demonstrated the presence of humoral immune responses to fungi, including Alternaria and Aspergillus, in patients with nasal polyposis, asthma, or rhinitis. The purpose of this study was to evaluate the role of Alternaria- and Aspergillus-specific IgE antibodies in allergic inflammation of adenoid tissue. Methods: Thirty-nine atopic subjects who were sensitized to more than one common aeroallergen and 39 non-atopic subjects undergoing adenotonsillectomy were recruited. The Phadia ImmunoCAP was used to quantify total IgE, Alternaria- and Aspergillus-specific IgE, eosinophil cationic protein (ECP), and mast cell tryptase in adenoid tissue homogenates. Alternaria- and Aspergillus-specific IgE were detected in the adenoid tissues from some of the subjects (37.2% and 24.4%, respectively) without systemic sensitization to common airborne fungi. Results: Both Alternaria- and Aspergillus-specific IgE were more prevalent in adenoid tissues from atopic children (48.7% and 38.5%, respectively) than in tissues from non-atopic children (25.6% and 10.3%, respectively). Subjects with high Alternaria-specific IgE level showed significantly higher serum and adenoid total IgE and adenoid ECP and tryptase than those without specific IgE. Alternaria-specific IgE levels were significantly correlated with serum and adenoid total IgE and with tryptase and ECP levels in adenoid tissue. Conclusions: Adenoid tissues from atopic and non-atopic children displayed local IgE-mediated hypersensitivity to fungi in the absence of systemic fungal hypersensitivity. Locally-produced Alternaria-specific IgE may contribute to mast cell and eosinophil activation, especially in the presence of tissue eosinophilia. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Alternaria-specific IgE Aspergillus-specific IgE Adenoid Eosinophil Mast cell
1. Introduction Fungi, ubiquitous in the environment, may coexist without effect in a host with normal cellular immunity [1] or may contribute to allergic airway diseases in those who are susceptible and/or heavily exposed [2–4]. Among the normal contaminants in nasal mucus, Alternaria alternata and Aspergillus fulmigatus are frequently detected [5], and patients with nasal polyposis, asthma and fungal sinusitis express humoral immune responses to such fungi [6–8]. The two chief effector cells in allergic response are the mast cells and the eosinophils. In the nasal and bronchial mucosa, allergic inflammation is characterized by tissue eosinophilia.
§ This study was supported by a grant by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST, 2009-0078646). * Corresponding author at: Department of Allergy and Clinical Immunology, Ajou University Scool of Medicine, Wonchondong San-5, Paldalgu, Suwon 442-749, Republic of Korea. Tel.: +82 31 219 5150; fax: +82 31 219 5154. E-mail address: [email protected] (H.S. Park).
0165-5876/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2012.06.015
According to a recent study, the innate direct response of eosinophils to certain fungi (e.g., Alternaria), as well as the humoral immune response, may play a prominent role in the pathophysiology and exacerbation of asthma and other eosinophilrelated airway diseases [9]. In previous studies, we demonstrated the local production of total IgE and allergen-specific IgE in adenoid tissues and the relationship between local allergen-specific IgE and allergic inflammatory mediators such as tryptase and ECP in adenoid tissues [10]. However, to the best of our knowledge, few studies have evaluated local IgE-mediated fungal hypersensitivity in tissues from pediatric patients. To confirm local production of Alternaria- and Aspergillus-specific IgE, we recruited pediatric patients with and without atopy. To evaluate the roles of Alternaria- and Aspergillus-specific IgE in allergic inflammation of adenoid tissue, we measured Alternaria- and Aspergillusspecific IgE levels in adenoid tissue homogenates from the two groups. We also measured the following parameters: serum total IgE; peripheral blood total eosinophil counts (TEC); and total IgE, tryptase, and ECP levels in adenoid tissue homogenates. We also
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observed the relationships between Alternaria- and Aspergillusspecific IgE and inflammatory markers within adenoid tissues.
system (Pharmacia, Uppsala, Sweden). An Alternaria- and/or Aspergillus-specific IgE level higher than 0.34 kUA/L was considered a positive result.
2. Materials and methods 2.1. Subjects Seventy-eight children (56 boys and 22 girls, mean age 7.5 years, range 3.2–13.5) participated in this study. All patients visited the ENT department in need of surgery for recurrent infections, including pharyngotonsillitis, rhinosinusitis, adenitis, and otitis media, or for problems related to adenotonsiller hypertrophy, including upper airway obstruction, sleep disorder, abnormal dentofacial growth, and dysphagia. The parents or foster parents of the children were asked to complete an extensive questionnaire on symptoms and signs of the child’s disease(s), medication, and family history with respect to allergy, rhinoconjunctivitis, asthma, eczema, smoking habits, and contact with pets. Before surgery, all children underwent a routine ENT examination. Children with congenital anomalies in the head and neck, Down syndrome, systemic diseases, or those suspected of having congenital or acquired immune deficiency were excluded from this study. Patients were also excluded if medication containing antihistamines were used intraoperatively or within the preceding week or if immunosuppressive agents, including steroids, were used intraoperatively or within the preceding six weeks. Blood samples obtained from patients before the operation were used for multiple allergosorbent chemiluminescence assay (MAST-CLA), to determine serum total IgE concentration using a BN II assay (Dade Behring, Marburg, Germany) and to count eosinophils using the ADVIA120 Hematology System (Bayer Diagnostics, NY, USA). For MAST-CLA, 40 antigens, including house dust mite, cockroach, cat, dog, tree pollen, grass pollen, weed pollen, and common airborne fungi, attached to threads were sequentially incubated with patient serum, enzyme-linked antiIgE, and a photoresponse reagent. The chemical fluorescence generated by each thread assay was developed on Polaroid film and scored according to a five-point system, from 0 to 4. Atopy status was defined as a total IgE higher than expected for age (>10 IU per year of age) and a score of more than 2+ on the MASTCLA. All subjects underwent adenotonsillectomy between May 2008 and July 2008 at the ENT Department of the Kyung Hee University Hospital in Seoul, Korea. Participation was subject to parental informed consent, and the study was approved by the local ethics committee.
2.4. Measurement of total IgE, ECP, and tryptase in adenoid tissue homogenate Total IgE was measured using the ImmunoCAP system (Pharmacia, Uppsala, Sweden), with the confidence range of 0.35–100 kUA/L. ECP and tryptase in adenoid homogenates diluted 1:5 and 1:20, respectively, were quantified by fluoroimmunoassay using the ImmunoCAP system (Pharmacia). The minimum detection limits for ECP and tryptase were 2 and 4 ng/mL, respectively. All parameters were corrected for albumin content and expressed as nanograms per milligram of albumin. 2.5. Statistical analyses The levels of Alternaria- and Aspergillus-specific IgE and inflammatory markers, including ECP and tryptase, were compared between atopic and non-atopic patients and between Alternariaspecific IgE-positive and negative patients using Mann–Whitney U-tests with SPSS version 12.0 (Chicago, IL, USA). Pearson’s correlation coefficients were applied to evaluate relationships between levels of Alternaria- and Aspergillus-specific IgE and inflammatory markers. P-values 0.05 or less were regarded as significant. 3. Results 3.1. Clinical characteristics of study subjects Thirty-eight patients (50%), including 29 boys and 10 girls, were found to have atopy, meaning they had excessively high total IgE for their age (>10 IU/year of age), and a score of more than 2+ in the MAST-CLA. Thirty-eight patients (50%) were non-atopic and served as a control group. The characteristics of the study subjects are shown in Table 1. The two groups did not differ significantly in age or sex (P > 0.05, respectively). Total IgE in sera and total eosinophil counts in peripheral blood of atopics were significantly higher than those of non-atopics (P = 0.016, P = 0.041, respectively). None of 78 children had specific IgE antibody to Alternaria alternata and Aspergillus fulmigatus in serum using MAST-CLA and ImmunoCAP.
2.2. Tissue collection Adenoidectomy was performed using a curette, and tissues were stored frozen after washing. Later, the washed adenoids were thawed and dispersed in LIPA buffer (Sigma–Aldrich, St. Louis, MO, USA) containing a protease inhibitor (Roche Diagnostics, Indianapolis, IN, USA). After centrifugation, the supernatants were collected and stored at 70 8C until use. All parameters were presented relative to the albumin level in adenoid tissue homogenates to correct for differences in the dilution factors between tissue samples [10]. Albumin levels in adenoid tissue homogenates were measured using a QuantiChromTM BCP Albumin Assay Kit (BioAssay Systems, Hayward, CA, USA). 2.3. Measurement of Alternaria- and Aspergillus-specific IgE in serum and adenoid tissue homogenates Alternaria- and Aspergillus-specific IgE were measured in serum and adenoid tissue homogenates using the ImmunoCAP
Table 1 Clinical data of the study subjects.
Gender (M/F) Age (years) Cormorbid diseases (positive/negative) Chronic rhinosinusitis Otitis media effusion Asthma Atopic dermatitis Serum total IgE (IU/mL) Adenoid total IgE/albumin Total eosinophil count (/mm3) in peripheral blood Tryptase/albumin in adenoid tissue ECP/albumin in adenoid tissue
Atopic (n = 39)
Non-atopic (n = 39) P
29/10 7.9 3.0
27/12 7.2 2.5
NS NS
16/23 6/33 1/37 3/34 333.44 543.84 22.25 20.58 255.49 150.36
21/18 8/31 2/38 4/37 99.17 110.91 8.80 9.14 189.56 125.28
NS NS NS NS 0.016 0.012 0.041
2794.69 2647.71 2696.72 2637.38
0.87
371.36 315.43
0.004
203.63 140.80
Values are expressed as mean SD. The Mann–Whitney U-test was used to compare atopic and non-atopic values.
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3.2. Comparison of total IgE, Alternaria- and Aspergillus-specific IgE, ECP, and tryptase in adenoid tissue homogenates between atopics and non-atopics Alternaria- and Aspergillus-specific IgE were detected in the adenoid tissues from some subjects (29 patients, 37.2% and 19 patients, 24.4%, respectively) (Fig. 1). The prevalences of Alternaria- and Aspergillus-specific IgE in adenoid tissues from atopic children (19 patients, 48.7% and 15 patients, 38.5%, respectively) were higher than those from non-atopic children (10 patients, 25.6% and 4 patients, 10.3%, respectively). All Aspergillus-positive children also presented positive responses to Alternaria. The levels of total IgE, ECP and tryptase in adenoid tissue homogenates from atopics and non-atopics are shown in Table 1. The total IgE and ECP levels in adenoid tissue were significantly higher in atopics than in non-atopics (P = 0.012, P = 0.004, respectively), but the two groups did not differ significantly in tryptase level (P = 0.87). 3.3. Stratification by Alternaria-specific IgE status The entire group of 78 children was stratified by the presence or absence of detectable Alternaria-specific IgE in adenoid tissues, irrespective of atopy status (Table 2). The subjects positive for Alternaria-specific IgE included some atopic (n = 19) and some non-atopic children (n = 10). Alternaria-positive and negative children did not differ in gender ratio or age. The Alternariapositive children had strikingly higher total IgE in adenoid tissue and sera (P < 0.01, both) and ECP and tryptase (P = 0.019, P = 0.007, respectively) than Alternaria-negative children (Table 2). Moreover, Alternaria-positive atopic children showed the highest level of ECP compared with Alternaria-positive non-atopic children, Alternaria-negative atopic children, and Alternaria-negative nonatopic children (Fig. 2). 3.4. Relationship between inflammatory markers and Alternaria- and Aspergillus-specific IgE levels in adenoid tissues Levels of Alternaria- and Aspergillus-specific IgE were significantly correlated with levels of serum total IgE (r = 0.463, r = 0.561,
[(Fig._1)TD$IG]
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respectively) and adenoid total IgE (r = 0.656, r = 0.653, respectively). In addition, levels of Alternaria-specific IgE were significantly correlated with levels of ECP and tryptase (r = 0.294, r = 0.343, respectively) in adenoid tissue homogenates, but Aspergillus-specific IgE correlated only weakly with ECP and tryptase (r = 0.184, r = 0.219, respectively) (Fig. 3).
4. Discussion In this study, we detected local IgE-mediated hypersensitivity to fungi in the adenoid tissues from both atopic and non-atopic children who did not show systemic sensitization to common airborne fungi. The adenoids with positive responses to Alternariaspecific IgE presented higher mast cell tryptase and ECP levels than those showing negative responses, and the level of Alternariaspecific IgE showed significant correlations with tryptase and ECP in adenoid tissues. Moreover, these responses were prominent in the adenoids from atopic children with hypersensitivity to Alternaria. The recognition of fungi as a causal factor in allergic airways diseases drew attention to the prevalence of fungal sensitization in young children [11,12]. A prevalence ranging from 30 to 40% was found, with an age-dependent distribution reaching maximum values at 7–8 years. Sensitization to Alternaria, Cladosporium, and Aspergillus is very common, as they are the fungi detected in greatest abundance in indoor and outdoor air [11,12]. Sensitization to Alternaria may predict the development of asthma [2] and appears to be a risk factor for respiratory arrest in patients with asthma [13]. In previous studies, we demonstrated a localized allergic response to inhalant allergens and microbial products within adenoid tissues [10,14]. The peak age of sensitization to fungi is generally similar to that of patients receiving adenotonsillectomy; and since the adenoid tissues, in contrast to nasal mucosa, are a suitable material for studying local allergic inflammation, we planned to evaluate the local IgE-mediated hypersensitivity to fungi in children. Considering the strong allergenicity of Alternaria and Aspergillus species and their close associations with upper and lower respiratory diseases, we used these common airborne fungi as the allergens for our study.
Fig. 1. Alternaria-specific IgE (*) and Aspergillus-specific IgE (~) levels in homogenates of adenoid tissue from atopic (n = 39) and non-atopic (n = 39) children. Groups were compared using the Mann–Whitney U-test, and dashed horizontal bars indicate the cut-off value for a positive response (0.34 kIUA/L). *<0.01.
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Table 2 Stratification of study subjects based on the presence or absence of Alternaria-specific IgE in adenoid tissue.
Gender (M/F) Age (years) Serum total IgE (IU/mL) Adenoid total IgE/albumin Total eosinophil count (/mm3) in peripheral blood Tryptase/albumin in adenoid ECP/albumin in adenoid
Alternaria-specific IgE positive (n = 29)
Alternaria-specific IgE negative (n = 49)
P
22/7 7.31 2.72 403.67 596.25 29.92 33.25 232.25 121.56 3646.56 3143.64 385.49 295.07
34/15 7.12 3.11 97.16 114.25 7.00 5.97 215.48 152.20 2212.54 2124.89 229.50 214.17
NS NS <0.001 <0.001 0.227 0.007 0.019
Values are expressed as mean SD. The Mann–Whitney U-test was used to compare atopic and non-atopic values.
In the present study, 29 of 78 children had demonstrable IgE antibody for Alternaria in adenoid tissue. Nineteen of these 29 children also had IgE for Aspergillus, which suggests that the prevalence of IgE for Alternaria is higher than that for Aspergillus (Fig. 1). None of these randomly selected 29 children had specific IgE antibody to the corresponding species in serum using MASTCLA and ImmunoCAP. These findings support the hypothesis that IgE-mediated fungal hypersensitivity may occur locally in adenoid tissues, without systemic sensitization. Interestingly, 10 of these 29 children were non-atopic, which suggested that local allergic inflammation may be less dependent than systemic inflammation on allergic predisposition. Previous studies detected multiclonal IgE production in some nasal polyps, in association with local IgE-mediated hypersensitivity to Alternaria or Staphylococcus [6,15,16]. Accordingly, Alternaria was recently proposed to be a superantigen comparable to staphylococcal superantigen [6]. In a previous study, we demonstrated multiclonal IgE production in some adenoid tissues, in association with staphylococcal superantigen [14]. In this present study, we detected multiclonal IgE production in some adenoid tissues that showed local IgE-mediated hypersensitivity to Alternaria (Table 2). From that, we concluded that adenoid tissues, like nasal polyps, may produce multiclonal IgE in association with local IgE-mediated hypersensitivity to Alternaria or Staphylococcus, and that Alternaria may act as a superantigen, in a manner similar to staphylococcal superantigen. The inconsistent point
[(Fig._2)TD$IG]
between our adenoid studies was that we detected local IgEmediated hypersensitivity to Staphylococcus only in atopic patients but local IgE-mediated hypersensitivity to Alternaria in both atopics and non-atopics. Mast cells are widely recognized as the principle effector cells in IgE-mediated allergic reactions following secondary contact with allergens. Tryptase is the most abundant secretory granule-derived serine proteinase contained in mast cells and is used as a marker for mast cell activation. However, few studies demonstrate an actual increase in tryptase expression or an obligate role for tryptase that is associated with allergen-specific IgE in tissues [16,17]. In previous studies, we also could not detect a significant difference in the level of tryptase in adenoid tissue homogenates between atopics and non-atopics, while the level of ECP showed a significant difference between the two groups at all times. In the present study, as in our previous one, tryptase levels in adenoid tissue homogenates from atopics and non-atopics did not differ significantly (Table 1). However, tryptase levels were higher in Alternaria-specific IgE-positive patients than in Alternaria-specific IgE-negative patients (Table 2). Moreover, tryptase levels showed a strong correlation with Alternaria-specific IgE levels in adenoid tissue homogenates (Fig. 3). These findings may explain in part the high capacity of Alternaria spores to sensitize tissues compared with other fungi and inhalant allergens, and the tissue-specific induction of tryptase may potentially predict the development of allergic diseases.
Fig. 2. ECP levels corrected for albumin in homogenates of adenoid tissue from Alternaria-specific IgE-positive atopic (n = 19), Alternaria-specific IgE-positive non-atopic (n = 10), Alternaria-specific IgE-negative atopic (n = 20) and Alternaria-specific IgE-negative non-atopic subjects (n = 29). Groups were compared using the Kruskal–Wallis and Mann–Whitney U-test, and horizontal bars indicate median values. *<0.05, **<0.01.
[(Fig._3)TD$IG]
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Fig. 3. Correlations between Alternaria-specific IgE, and serum total IgE (A), adenoid total IgE (B), tryptase (C), and ECP (D) levels in adenoid tissue homogenates from the study subjects.
Eosinophils are implicated in the pathophysiology of allergic diseases and in host immunity to certain organisms. Moreover, tissue eosinophilia is a hallmark of severity in eosinophil-related upper and lower airway diseases such as allergic rhinitis, nasal polyp, and asthma. Inoue et al. demonstrated that nonpathogenic, environmental fungi, including Alternaria and Penicillium, can induce eosinophil activation and degranulation, and suggested that the innate response by eosinophils to certain environmental fungi plays an important role in asthma and allergic diseases [9]. In the present study, ECP levels differed significantly between atopic and non-atopic subjects, in concordance with our previous studies (Table 1). The ECP levels also differed significantly between Alternaria-specific IgE-positive and negative patients (Table 2) and showed a significant correlation with Alternaria-specific IgE level in adenoid tissue homogenates (Fig. 3). Accordingly, we further divided the subjects into four groups according to the presence of atopy and positive response to Alternaria-specific IgE and compared the ECP levels among them. Interestingly, atopics with positive response for Alternaria-specific IgE showed the highest ECP levels, and these differed significantly from those of the other three groups (Fig. 2). We thus concluded that Alternaria spores may have the capacity to trigger eosinophil activation and degranulation, especially in the condition of tissue eosinophilia induced by allergic response to inhalant allergens, and may serve
as a predictive factor for the development or exacerbation of allergic diseases. 5. Conclusions Local IgE-mediated fungal hypersensitivity was confirmed in the adenoid tissues of atopic and non-atopic children who did not show systemic fungal hypersensitivity. Locally-produced Alternaria-specific IgE may contribute to mast cell and eosinophil activation, especially in the condition of tissue eosinophilia. Therefore, we have to look for possible local production of IgE to fungus in childhood patients with persistent inflammation composing eosinophil and mast cell in adenoid tissue. References [1] J.A. van Burik, P.T. Magee, Aspects of fungal pathogenesis in humans, Annu. Rev. Microbiol. 55 (2001) 743–772. [2] R.K. Bush, J.J. Prochnau, Alternaria-induced asthma, J. Allergy Clin. Immunol. 113 (2004) 227–234. [3] H.F. Kauffman, J.F. Tomee, T.S. van der Werf, J.G. de Monchy, G.K. Koe¨ter, Review of fungus-induced asthmatic reactions, Am. J. Respir. Crit. Care Med. 151 (1995) 2109–2115. [4] J.F. Tomee, H.F. Kauffman, Putative virulence factors of Aspergillus fumigatus, Clin. Exp. Allergy 30 (2000) 476–484.
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[5] L.J. Davis, H. Kita, Pathogenesis of chronic rhinosinusitis: role of airborne fungi and bacteria, Immunol. Allergy Clin. North Am. 24 (2004) 59–73. [6] A. Sabirov, R.G. Hamilton, J.B. Jacobs, D.E. Hillman, R.A. Lebowitz, J.D. Watts, Role of local immunoglobulin E specific for Alternaria alternata in the pathogenesis of nasal polyposis, Laryngoscope 118 (2008) 4–9. [7] R. Crameri, A. Faith, S. Hemmann, R. Jaussi, C. Ismail, G. Menz, et al., Humoral and cell-mediated autoimmunity in allergy to Aspergillus fumigatus, J. Exp. Med. 184 (1996) 265–270. [8] H. Pant, F.E. Kette, W.B. Smith, P.J. Wormald, P.J. Macardle, Fungal-specific humoral response in eosinophilic mucus chronic rhinosinusitis, Laryngoscope 115 (2005) 601–606. [9] Y. Inoue, Y. Matsuwaki, S.H. Shin, J.U. Ponikau, H. Kita, Nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils, J. Immunol. 175 (2005) 5439–5447. [10] S.Y. Shin, S.J. Choi, G.Y. Hur, K.H. Lee, S.W. Kim, J.S. Cho, et al., Local production of total IgE and specific antibodies to the house dust mite in adenoid tissue, Pediatr. Allergy Immunol. 20 (2009) 134–141. [11] G. Nolles, M.O. Hoekstra, J.P. Schouten, J. Gerritsen, H.F. Kauffman, Prevalence of immunoglobulin E for fungi in atopic children, Clin. Exp. Allergy 31 (2001) 1564– 1570.
[12] National Heart, Lung and Blood Institute, National Institutes of Health, International consensus report on diagnosis and treatment of asthma, Publication no. 923091, Eur. Respir. J. 5 (1992) 601–641. [13] C. Neukirch, C. Henry, B. Leynaert, R. Liard, J. Bousquet, F. Neukirch, Is sensitization to Alternaria a risk factor for severe asthma? A population based study, J. Allergy Clin. Immunol. 103 (1999) 709–711. [14] S.Y. Shin, G.S. Choi, K.H. Lee, S.W. Kim, J.S. Cho, H.S. Park, IgE response to staphylococcal enterotoxins in adenoid tissues from atopic children, Laryngoscope 119 (2009) 171–175. [15] P. Gevaert, G. Holtappels, S.G. Johansson, C. Cuvelier, P. Cauwenberge, C. Bachert, Organization of secondary lymphoid tissue and local IgE formation to Staphylococcus aureus enterotoxins in nasal polyp tissue, Allergy 60 (2005) 71–79. [16] C. Bachert, P. Gevaert, G. Holtappels, S.G. Johansson, P. van Cauwenberge, Total and specific IgE in nasal polyps is related to local eosinophilic inflammation, J. Allergy Clin. Immunol. 107 (2001) 607–614. [17] Y.J. Suh, S.H. Yoon, A.P. Sampson, H.J. Kim, S.H. Kim, D.H. Nahm, et al., Specific immunoglobulin E for staphylococcal enterotoxins in nasal polyps from patients with aspirin-intolerant asthma, Clin. Exp. Allergy 34 (2004) 1270–1275.
Contents lists available at SciVerse ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Local IgE-mediated hypersensitivity to Alternaria in pediatric adenoid tissue§ Seung Youp Shin a, Young Min Ye b, Young Gyu Eun a, Sung Wan Kim a, Joong Saeng Cho a, Hae Sim Park b,* a b
Department of Otorhinolaryngology-Head and Neck Surgery, Kyung Hee University School of Medicine, Seoul, Republic of Korea Department of Allergy and Clinical Immunology, Ajou University School of Medicine, Suwon, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 October 2011 Received in revised form 13 June 2012 Accepted 14 June 2012 Available online 6 July 2012
Objective: Fungus may contribute to the development and exacerbation of allergic airway diseases. Several studies have demonstrated the presence of humoral immune responses to fungi, including Alternaria and Aspergillus, in patients with nasal polyposis, asthma, or rhinitis. The purpose of this study was to evaluate the role of Alternaria- and Aspergillus-specific IgE antibodies in allergic inflammation of adenoid tissue. Methods: Thirty-nine atopic subjects who were sensitized to more than one common aeroallergen and 39 non-atopic subjects undergoing adenotonsillectomy were recruited. The Phadia ImmunoCAP was used to quantify total IgE, Alternaria- and Aspergillus-specific IgE, eosinophil cationic protein (ECP), and mast cell tryptase in adenoid tissue homogenates. Alternaria- and Aspergillus-specific IgE were detected in the adenoid tissues from some of the subjects (37.2% and 24.4%, respectively) without systemic sensitization to common airborne fungi. Results: Both Alternaria- and Aspergillus-specific IgE were more prevalent in adenoid tissues from atopic children (48.7% and 38.5%, respectively) than in tissues from non-atopic children (25.6% and 10.3%, respectively). Subjects with high Alternaria-specific IgE level showed significantly higher serum and adenoid total IgE and adenoid ECP and tryptase than those without specific IgE. Alternaria-specific IgE levels were significantly correlated with serum and adenoid total IgE and with tryptase and ECP levels in adenoid tissue. Conclusions: Adenoid tissues from atopic and non-atopic children displayed local IgE-mediated hypersensitivity to fungi in the absence of systemic fungal hypersensitivity. Locally-produced Alternaria-specific IgE may contribute to mast cell and eosinophil activation, especially in the presence of tissue eosinophilia. ß 2012 Elsevier Ireland Ltd. All rights reserved.
Keywords: Alternaria-specific IgE Aspergillus-specific IgE Adenoid Eosinophil Mast cell
1. Introduction Fungi, ubiquitous in the environment, may coexist without effect in a host with normal cellular immunity [1] or may contribute to allergic airway diseases in those who are susceptible and/or heavily exposed [2–4]. Among the normal contaminants in nasal mucus, Alternaria alternata and Aspergillus fulmigatus are frequently detected [5], and patients with nasal polyposis, asthma and fungal sinusitis express humoral immune responses to such fungi [6–8]. The two chief effector cells in allergic response are the mast cells and the eosinophils. In the nasal and bronchial mucosa, allergic inflammation is characterized by tissue eosinophilia.
§ This study was supported by a grant by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST, 2009-0078646). * Corresponding author at: Department of Allergy and Clinical Immunology, Ajou University Scool of Medicine, Wonchondong San-5, Paldalgu, Suwon 442-749, Republic of Korea. Tel.: +82 31 219 5150; fax: +82 31 219 5154. E-mail address: [email protected] (H.S. Park).
0165-5876/$ – see front matter ß 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijporl.2012.06.015
According to a recent study, the innate direct response of eosinophils to certain fungi (e.g., Alternaria), as well as the humoral immune response, may play a prominent role in the pathophysiology and exacerbation of asthma and other eosinophilrelated airway diseases [9]. In previous studies, we demonstrated the local production of total IgE and allergen-specific IgE in adenoid tissues and the relationship between local allergen-specific IgE and allergic inflammatory mediators such as tryptase and ECP in adenoid tissues [10]. However, to the best of our knowledge, few studies have evaluated local IgE-mediated fungal hypersensitivity in tissues from pediatric patients. To confirm local production of Alternaria- and Aspergillus-specific IgE, we recruited pediatric patients with and without atopy. To evaluate the roles of Alternaria- and Aspergillus-specific IgE in allergic inflammation of adenoid tissue, we measured Alternaria- and Aspergillusspecific IgE levels in adenoid tissue homogenates from the two groups. We also measured the following parameters: serum total IgE; peripheral blood total eosinophil counts (TEC); and total IgE, tryptase, and ECP levels in adenoid tissue homogenates. We also
1424
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observed the relationships between Alternaria- and Aspergillusspecific IgE and inflammatory markers within adenoid tissues.
system (Pharmacia, Uppsala, Sweden). An Alternaria- and/or Aspergillus-specific IgE level higher than 0.34 kUA/L was considered a positive result.
2. Materials and methods 2.1. Subjects Seventy-eight children (56 boys and 22 girls, mean age 7.5 years, range 3.2–13.5) participated in this study. All patients visited the ENT department in need of surgery for recurrent infections, including pharyngotonsillitis, rhinosinusitis, adenitis, and otitis media, or for problems related to adenotonsiller hypertrophy, including upper airway obstruction, sleep disorder, abnormal dentofacial growth, and dysphagia. The parents or foster parents of the children were asked to complete an extensive questionnaire on symptoms and signs of the child’s disease(s), medication, and family history with respect to allergy, rhinoconjunctivitis, asthma, eczema, smoking habits, and contact with pets. Before surgery, all children underwent a routine ENT examination. Children with congenital anomalies in the head and neck, Down syndrome, systemic diseases, or those suspected of having congenital or acquired immune deficiency were excluded from this study. Patients were also excluded if medication containing antihistamines were used intraoperatively or within the preceding week or if immunosuppressive agents, including steroids, were used intraoperatively or within the preceding six weeks. Blood samples obtained from patients before the operation were used for multiple allergosorbent chemiluminescence assay (MAST-CLA), to determine serum total IgE concentration using a BN II assay (Dade Behring, Marburg, Germany) and to count eosinophils using the ADVIA120 Hematology System (Bayer Diagnostics, NY, USA). For MAST-CLA, 40 antigens, including house dust mite, cockroach, cat, dog, tree pollen, grass pollen, weed pollen, and common airborne fungi, attached to threads were sequentially incubated with patient serum, enzyme-linked antiIgE, and a photoresponse reagent. The chemical fluorescence generated by each thread assay was developed on Polaroid film and scored according to a five-point system, from 0 to 4. Atopy status was defined as a total IgE higher than expected for age (>10 IU per year of age) and a score of more than 2+ on the MASTCLA. All subjects underwent adenotonsillectomy between May 2008 and July 2008 at the ENT Department of the Kyung Hee University Hospital in Seoul, Korea. Participation was subject to parental informed consent, and the study was approved by the local ethics committee.
2.4. Measurement of total IgE, ECP, and tryptase in adenoid tissue homogenate Total IgE was measured using the ImmunoCAP system (Pharmacia, Uppsala, Sweden), with the confidence range of 0.35–100 kUA/L. ECP and tryptase in adenoid homogenates diluted 1:5 and 1:20, respectively, were quantified by fluoroimmunoassay using the ImmunoCAP system (Pharmacia). The minimum detection limits for ECP and tryptase were 2 and 4 ng/mL, respectively. All parameters were corrected for albumin content and expressed as nanograms per milligram of albumin. 2.5. Statistical analyses The levels of Alternaria- and Aspergillus-specific IgE and inflammatory markers, including ECP and tryptase, were compared between atopic and non-atopic patients and between Alternariaspecific IgE-positive and negative patients using Mann–Whitney U-tests with SPSS version 12.0 (Chicago, IL, USA). Pearson’s correlation coefficients were applied to evaluate relationships between levels of Alternaria- and Aspergillus-specific IgE and inflammatory markers. P-values 0.05 or less were regarded as significant. 3. Results 3.1. Clinical characteristics of study subjects Thirty-eight patients (50%), including 29 boys and 10 girls, were found to have atopy, meaning they had excessively high total IgE for their age (>10 IU/year of age), and a score of more than 2+ in the MAST-CLA. Thirty-eight patients (50%) were non-atopic and served as a control group. The characteristics of the study subjects are shown in Table 1. The two groups did not differ significantly in age or sex (P > 0.05, respectively). Total IgE in sera and total eosinophil counts in peripheral blood of atopics were significantly higher than those of non-atopics (P = 0.016, P = 0.041, respectively). None of 78 children had specific IgE antibody to Alternaria alternata and Aspergillus fulmigatus in serum using MAST-CLA and ImmunoCAP.
2.2. Tissue collection Adenoidectomy was performed using a curette, and tissues were stored frozen after washing. Later, the washed adenoids were thawed and dispersed in LIPA buffer (Sigma–Aldrich, St. Louis, MO, USA) containing a protease inhibitor (Roche Diagnostics, Indianapolis, IN, USA). After centrifugation, the supernatants were collected and stored at 70 8C until use. All parameters were presented relative to the albumin level in adenoid tissue homogenates to correct for differences in the dilution factors between tissue samples [10]. Albumin levels in adenoid tissue homogenates were measured using a QuantiChromTM BCP Albumin Assay Kit (BioAssay Systems, Hayward, CA, USA). 2.3. Measurement of Alternaria- and Aspergillus-specific IgE in serum and adenoid tissue homogenates Alternaria- and Aspergillus-specific IgE were measured in serum and adenoid tissue homogenates using the ImmunoCAP
Table 1 Clinical data of the study subjects.
Gender (M/F) Age (years) Cormorbid diseases (positive/negative) Chronic rhinosinusitis Otitis media effusion Asthma Atopic dermatitis Serum total IgE (IU/mL) Adenoid total IgE/albumin Total eosinophil count (/mm3) in peripheral blood Tryptase/albumin in adenoid tissue ECP/albumin in adenoid tissue
Atopic (n = 39)
Non-atopic (n = 39) P
29/10 7.9 3.0
27/12 7.2 2.5
NS NS
16/23 6/33 1/37 3/34 333.44 543.84 22.25 20.58 255.49 150.36
21/18 8/31 2/38 4/37 99.17 110.91 8.80 9.14 189.56 125.28
NS NS NS NS 0.016 0.012 0.041
2794.69 2647.71 2696.72 2637.38
0.87
371.36 315.43
0.004
203.63 140.80
Values are expressed as mean SD. The Mann–Whitney U-test was used to compare atopic and non-atopic values.
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3.2. Comparison of total IgE, Alternaria- and Aspergillus-specific IgE, ECP, and tryptase in adenoid tissue homogenates between atopics and non-atopics Alternaria- and Aspergillus-specific IgE were detected in the adenoid tissues from some subjects (29 patients, 37.2% and 19 patients, 24.4%, respectively) (Fig. 1). The prevalences of Alternaria- and Aspergillus-specific IgE in adenoid tissues from atopic children (19 patients, 48.7% and 15 patients, 38.5%, respectively) were higher than those from non-atopic children (10 patients, 25.6% and 4 patients, 10.3%, respectively). All Aspergillus-positive children also presented positive responses to Alternaria. The levels of total IgE, ECP and tryptase in adenoid tissue homogenates from atopics and non-atopics are shown in Table 1. The total IgE and ECP levels in adenoid tissue were significantly higher in atopics than in non-atopics (P = 0.012, P = 0.004, respectively), but the two groups did not differ significantly in tryptase level (P = 0.87). 3.3. Stratification by Alternaria-specific IgE status The entire group of 78 children was stratified by the presence or absence of detectable Alternaria-specific IgE in adenoid tissues, irrespective of atopy status (Table 2). The subjects positive for Alternaria-specific IgE included some atopic (n = 19) and some non-atopic children (n = 10). Alternaria-positive and negative children did not differ in gender ratio or age. The Alternariapositive children had strikingly higher total IgE in adenoid tissue and sera (P < 0.01, both) and ECP and tryptase (P = 0.019, P = 0.007, respectively) than Alternaria-negative children (Table 2). Moreover, Alternaria-positive atopic children showed the highest level of ECP compared with Alternaria-positive non-atopic children, Alternaria-negative atopic children, and Alternaria-negative nonatopic children (Fig. 2). 3.4. Relationship between inflammatory markers and Alternaria- and Aspergillus-specific IgE levels in adenoid tissues Levels of Alternaria- and Aspergillus-specific IgE were significantly correlated with levels of serum total IgE (r = 0.463, r = 0.561,
[(Fig._1)TD$IG]
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respectively) and adenoid total IgE (r = 0.656, r = 0.653, respectively). In addition, levels of Alternaria-specific IgE were significantly correlated with levels of ECP and tryptase (r = 0.294, r = 0.343, respectively) in adenoid tissue homogenates, but Aspergillus-specific IgE correlated only weakly with ECP and tryptase (r = 0.184, r = 0.219, respectively) (Fig. 3).
4. Discussion In this study, we detected local IgE-mediated hypersensitivity to fungi in the adenoid tissues from both atopic and non-atopic children who did not show systemic sensitization to common airborne fungi. The adenoids with positive responses to Alternariaspecific IgE presented higher mast cell tryptase and ECP levels than those showing negative responses, and the level of Alternariaspecific IgE showed significant correlations with tryptase and ECP in adenoid tissues. Moreover, these responses were prominent in the adenoids from atopic children with hypersensitivity to Alternaria. The recognition of fungi as a causal factor in allergic airways diseases drew attention to the prevalence of fungal sensitization in young children [11,12]. A prevalence ranging from 30 to 40% was found, with an age-dependent distribution reaching maximum values at 7–8 years. Sensitization to Alternaria, Cladosporium, and Aspergillus is very common, as they are the fungi detected in greatest abundance in indoor and outdoor air [11,12]. Sensitization to Alternaria may predict the development of asthma [2] and appears to be a risk factor for respiratory arrest in patients with asthma [13]. In previous studies, we demonstrated a localized allergic response to inhalant allergens and microbial products within adenoid tissues [10,14]. The peak age of sensitization to fungi is generally similar to that of patients receiving adenotonsillectomy; and since the adenoid tissues, in contrast to nasal mucosa, are a suitable material for studying local allergic inflammation, we planned to evaluate the local IgE-mediated hypersensitivity to fungi in children. Considering the strong allergenicity of Alternaria and Aspergillus species and their close associations with upper and lower respiratory diseases, we used these common airborne fungi as the allergens for our study.
Fig. 1. Alternaria-specific IgE (*) and Aspergillus-specific IgE (~) levels in homogenates of adenoid tissue from atopic (n = 39) and non-atopic (n = 39) children. Groups were compared using the Mann–Whitney U-test, and dashed horizontal bars indicate the cut-off value for a positive response (0.34 kIUA/L). *<0.01.
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Table 2 Stratification of study subjects based on the presence or absence of Alternaria-specific IgE in adenoid tissue.
Gender (M/F) Age (years) Serum total IgE (IU/mL) Adenoid total IgE/albumin Total eosinophil count (/mm3) in peripheral blood Tryptase/albumin in adenoid ECP/albumin in adenoid
Alternaria-specific IgE positive (n = 29)
Alternaria-specific IgE negative (n = 49)
P
22/7 7.31 2.72 403.67 596.25 29.92 33.25 232.25 121.56 3646.56 3143.64 385.49 295.07
34/15 7.12 3.11 97.16 114.25 7.00 5.97 215.48 152.20 2212.54 2124.89 229.50 214.17
NS NS <0.001 <0.001 0.227 0.007 0.019
Values are expressed as mean SD. The Mann–Whitney U-test was used to compare atopic and non-atopic values.
In the present study, 29 of 78 children had demonstrable IgE antibody for Alternaria in adenoid tissue. Nineteen of these 29 children also had IgE for Aspergillus, which suggests that the prevalence of IgE for Alternaria is higher than that for Aspergillus (Fig. 1). None of these randomly selected 29 children had specific IgE antibody to the corresponding species in serum using MASTCLA and ImmunoCAP. These findings support the hypothesis that IgE-mediated fungal hypersensitivity may occur locally in adenoid tissues, without systemic sensitization. Interestingly, 10 of these 29 children were non-atopic, which suggested that local allergic inflammation may be less dependent than systemic inflammation on allergic predisposition. Previous studies detected multiclonal IgE production in some nasal polyps, in association with local IgE-mediated hypersensitivity to Alternaria or Staphylococcus [6,15,16]. Accordingly, Alternaria was recently proposed to be a superantigen comparable to staphylococcal superantigen [6]. In a previous study, we demonstrated multiclonal IgE production in some adenoid tissues, in association with staphylococcal superantigen [14]. In this present study, we detected multiclonal IgE production in some adenoid tissues that showed local IgE-mediated hypersensitivity to Alternaria (Table 2). From that, we concluded that adenoid tissues, like nasal polyps, may produce multiclonal IgE in association with local IgE-mediated hypersensitivity to Alternaria or Staphylococcus, and that Alternaria may act as a superantigen, in a manner similar to staphylococcal superantigen. The inconsistent point
[(Fig._2)TD$IG]
between our adenoid studies was that we detected local IgEmediated hypersensitivity to Staphylococcus only in atopic patients but local IgE-mediated hypersensitivity to Alternaria in both atopics and non-atopics. Mast cells are widely recognized as the principle effector cells in IgE-mediated allergic reactions following secondary contact with allergens. Tryptase is the most abundant secretory granule-derived serine proteinase contained in mast cells and is used as a marker for mast cell activation. However, few studies demonstrate an actual increase in tryptase expression or an obligate role for tryptase that is associated with allergen-specific IgE in tissues [16,17]. In previous studies, we also could not detect a significant difference in the level of tryptase in adenoid tissue homogenates between atopics and non-atopics, while the level of ECP showed a significant difference between the two groups at all times. In the present study, as in our previous one, tryptase levels in adenoid tissue homogenates from atopics and non-atopics did not differ significantly (Table 1). However, tryptase levels were higher in Alternaria-specific IgE-positive patients than in Alternaria-specific IgE-negative patients (Table 2). Moreover, tryptase levels showed a strong correlation with Alternaria-specific IgE levels in adenoid tissue homogenates (Fig. 3). These findings may explain in part the high capacity of Alternaria spores to sensitize tissues compared with other fungi and inhalant allergens, and the tissue-specific induction of tryptase may potentially predict the development of allergic diseases.
Fig. 2. ECP levels corrected for albumin in homogenates of adenoid tissue from Alternaria-specific IgE-positive atopic (n = 19), Alternaria-specific IgE-positive non-atopic (n = 10), Alternaria-specific IgE-negative atopic (n = 20) and Alternaria-specific IgE-negative non-atopic subjects (n = 29). Groups were compared using the Kruskal–Wallis and Mann–Whitney U-test, and horizontal bars indicate median values. *<0.05, **<0.01.
[(Fig._3)TD$IG]
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Fig. 3. Correlations between Alternaria-specific IgE, and serum total IgE (A), adenoid total IgE (B), tryptase (C), and ECP (D) levels in adenoid tissue homogenates from the study subjects.
Eosinophils are implicated in the pathophysiology of allergic diseases and in host immunity to certain organisms. Moreover, tissue eosinophilia is a hallmark of severity in eosinophil-related upper and lower airway diseases such as allergic rhinitis, nasal polyp, and asthma. Inoue et al. demonstrated that nonpathogenic, environmental fungi, including Alternaria and Penicillium, can induce eosinophil activation and degranulation, and suggested that the innate response by eosinophils to certain environmental fungi plays an important role in asthma and allergic diseases [9]. In the present study, ECP levels differed significantly between atopic and non-atopic subjects, in concordance with our previous studies (Table 1). The ECP levels also differed significantly between Alternaria-specific IgE-positive and negative patients (Table 2) and showed a significant correlation with Alternaria-specific IgE level in adenoid tissue homogenates (Fig. 3). Accordingly, we further divided the subjects into four groups according to the presence of atopy and positive response to Alternaria-specific IgE and compared the ECP levels among them. Interestingly, atopics with positive response for Alternaria-specific IgE showed the highest ECP levels, and these differed significantly from those of the other three groups (Fig. 2). We thus concluded that Alternaria spores may have the capacity to trigger eosinophil activation and degranulation, especially in the condition of tissue eosinophilia induced by allergic response to inhalant allergens, and may serve
as a predictive factor for the development or exacerbation of allergic diseases. 5. Conclusions Local IgE-mediated fungal hypersensitivity was confirmed in the adenoid tissues of atopic and non-atopic children who did not show systemic fungal hypersensitivity. Locally-produced Alternaria-specific IgE may contribute to mast cell and eosinophil activation, especially in the condition of tissue eosinophilia. Therefore, we have to look for possible local production of IgE to fungus in childhood patients with persistent inflammation composing eosinophil and mast cell in adenoid tissue. References [1] J.A. van Burik, P.T. Magee, Aspects of fungal pathogenesis in humans, Annu. Rev. Microbiol. 55 (2001) 743–772. [2] R.K. Bush, J.J. Prochnau, Alternaria-induced asthma, J. Allergy Clin. Immunol. 113 (2004) 227–234. [3] H.F. Kauffman, J.F. Tomee, T.S. van der Werf, J.G. de Monchy, G.K. Koe¨ter, Review of fungus-induced asthmatic reactions, Am. J. Respir. Crit. Care Med. 151 (1995) 2109–2115. [4] J.F. Tomee, H.F. Kauffman, Putative virulence factors of Aspergillus fumigatus, Clin. Exp. Allergy 30 (2000) 476–484.
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[5] L.J. Davis, H. Kita, Pathogenesis of chronic rhinosinusitis: role of airborne fungi and bacteria, Immunol. Allergy Clin. North Am. 24 (2004) 59–73. [6] A. Sabirov, R.G. Hamilton, J.B. Jacobs, D.E. Hillman, R.A. Lebowitz, J.D. Watts, Role of local immunoglobulin E specific for Alternaria alternata in the pathogenesis of nasal polyposis, Laryngoscope 118 (2008) 4–9. [7] R. Crameri, A. Faith, S. Hemmann, R. Jaussi, C. Ismail, G. Menz, et al., Humoral and cell-mediated autoimmunity in allergy to Aspergillus fumigatus, J. Exp. Med. 184 (1996) 265–270. [8] H. Pant, F.E. Kette, W.B. Smith, P.J. Wormald, P.J. Macardle, Fungal-specific humoral response in eosinophilic mucus chronic rhinosinusitis, Laryngoscope 115 (2005) 601–606. [9] Y. Inoue, Y. Matsuwaki, S.H. Shin, J.U. Ponikau, H. Kita, Nonpathogenic, environmental fungi induce activation and degranulation of human eosinophils, J. Immunol. 175 (2005) 5439–5447. [10] S.Y. Shin, S.J. Choi, G.Y. Hur, K.H. Lee, S.W. Kim, J.S. Cho, et al., Local production of total IgE and specific antibodies to the house dust mite in adenoid tissue, Pediatr. Allergy Immunol. 20 (2009) 134–141. [11] G. Nolles, M.O. Hoekstra, J.P. Schouten, J. Gerritsen, H.F. Kauffman, Prevalence of immunoglobulin E for fungi in atopic children, Clin. Exp. Allergy 31 (2001) 1564– 1570.
[12] National Heart, Lung and Blood Institute, National Institutes of Health, International consensus report on diagnosis and treatment of asthma, Publication no. 923091, Eur. Respir. J. 5 (1992) 601–641. [13] C. Neukirch, C. Henry, B. Leynaert, R. Liard, J. Bousquet, F. Neukirch, Is sensitization to Alternaria a risk factor for severe asthma? A population based study, J. Allergy Clin. Immunol. 103 (1999) 709–711. [14] S.Y. Shin, G.S. Choi, K.H. Lee, S.W. Kim, J.S. Cho, H.S. Park, IgE response to staphylococcal enterotoxins in adenoid tissues from atopic children, Laryngoscope 119 (2009) 171–175. [15] P. Gevaert, G. Holtappels, S.G. Johansson, C. Cuvelier, P. Cauwenberge, C. Bachert, Organization of secondary lymphoid tissue and local IgE formation to Staphylococcus aureus enterotoxins in nasal polyp tissue, Allergy 60 (2005) 71–79. [16] C. Bachert, P. Gevaert, G. Holtappels, S.G. Johansson, P. van Cauwenberge, Total and specific IgE in nasal polyps is related to local eosinophilic inflammation, J. Allergy Clin. Immunol. 107 (2001) 607–614. [17] Y.J. Suh, S.H. Yoon, A.P. Sampson, H.J. Kim, S.H. Kim, D.H. Nahm, et al., Specific immunoglobulin E for staphylococcal enterotoxins in nasal polyps from patients with aspirin-intolerant asthma, Clin. Exp. Allergy 34 (2004) 1270–1275.