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Released intranasal eosinophilic major basic protein as a diagnostic marker for polypoid chronic rhinosinusitis
Otolaryngology–Head and Neck Surgery (2010) 143, 386-391
ORIGINAL RESEARCH–ALLERGY
Released intranasal eosinophilic major basic protein as a diagnostic marker for polypoid chronic rhinosinusitis Christoph Schmid, MD, Walter Habermann, MD, Hannes Braun, MD, Markus Gugatschka, MD, Brad S. Oriel, Janel A. Smietana, and Heinz Stammberger, MD, Graz, Austria; and Buffalo, NY No sponsorships or competing interests have been disclosed for this article. ABSTRACT OBJECTIVE: To investigate the hypothesis that eosinophil major basic protein is released in high concentrations in the nasal mucus of patients with polypoid chronic rhinosinusitis. STUDY DESIGN: Single center, open, prospective trial. SETTING: Medical University of Graz, Austria. SUBJECTS AND METHODS: Patients with polypoid chronic rhinosinusitis (n ⫽ 23) were compared to three different control groups: patients with chronic rhinosinusitis-like symptoms but without general mucosal thickening, patients who underwent functional endoscopic sinus surgery for reasons other than chronic rhinosinusitis, and patients without sinus disease (total n ⫽ 21). Mucus was harvested from each patient using a standardized technique and analyzed for eosinophil major basic protein with an enzyme-linked immunosorbent assay. RESULTS: In the patient group with polypoid chronic rhinosinusitis, 20 of 23 (87%) were positive for eosinophil major basic protein. In contrast, only one control patient was positive, whereas the remainder had no detectable amount of eosinophil major basic protein in the mucus (P ⬍ 0.001 vs chronic rhinosinusitis). CONCLUSION: Toxic eosinophil major basic protein levels are elevated in polypoid chronic rhinosinusitis patients compared to control groups that have similar clinical presentations but upon closer examination turn out not to have chronic rhinosinusitis. In the future, the detection of eosinophil major basic protein in nasal mucus may become a sensitive and specific marker for chronic rhinosinusitis and a helpful diagnostic tool. © 2010 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
C
hronic sinusitis afflicts many individuals worldwide. The 2006 National Health Interview Survey for the civilian noninstitutionalized adult population of the United States found the prevalence of chronic sinusitis in the adult population to be 13.8 percent, making it one of the most
common chronic diseases.1 Additionally, the German Society for Otorhinolaryngology–Head and Neck Surgery reported that the diagnosis of chronic sinusitis was made 2.6 million times in Germany in 2002, which resulted in 3.4 million medical prescriptions.2 Currently, controversy exists over the etiology and pathogenesis of chronic rhinosinusitis (CRS).3 Two hypothesized immunologic mechanisms are discussed below. Some authors propose that Staphylococcus aureus enterotoxins are implicated in the pathophysiology of CRS. These enterotoxins function as superantigens, resulting in local production of polyclonal immunoglobulin E, which may contribute to the severe eosinophilic inflammation mediated primarily by mast cells.4 According to the second hypothesis, most CRS is caused by a pathologic immune response to airborne fungi.5 These fungi are ubiquitous and can be detected in the nasal mucus of more than 90 percent of CRS patients as well as healthy subjects.5,6 However, only CRS patients produce interleukin (IL)-5 and IL-13 in response to the fungi, which are crucial cytokines known to drive the eosinophilic inflammation in CRS.7 The eosinophils do not remain in the tissue but actively move to the mucus, where they form clusters around the fungi.5 Regardless of the immunological mechanism proposed, both lead to an eosinophilic-dominated inflammation. Thus, the postulated damage of the tissue by toxic granular proteins of eosinophilic granulocytes may be valid for both theories. Eosinophilic granulocytes contain four principal cytotoxic proteins in their granules. The granular matrix is composed of eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). The characteristic granule core consists of eosinophil major basic protein (eMBP). EPO constitutes about 25 percent of the total protein mass of eosinophil granules.8,9 In the presence of hydrogen peroxide, EPO functions as a peroxidase with strong helminthotoxic activities, but it is also able to damage respiratory epithelium. EDN has mul-
Received September 16, 2009; revised May 11, 2010; accepted May 14, 2010.
0194-5998/$36.00 © 2010 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2010.05.017
Schmid et al
Released intranasal eosinophilic major basic protein . . .
tiple functions. It is a powerful neurotoxin, a potent ribonuclease, and possesses weak helminthotoxic activities.8,9 ECP, like EDN, is a member of the ribonuclease gene superfamily. Although ECP is toxic to bacteria, helminthes, protozoa, and mammalian cells, it also has a number of additional noncytotoxic effects, including suppression of T-cell responses, induction of mast cell degranulation, and stimulation of airway mucus secretion.8,9 eMBP is the principle protein in the eosinophil granule, comprising roughly 50 percent of the total granule protein mass, and has been found at concentrations exceeding more than a thousand times the amount needed to damage epithelium in the nasal mucus of CRS patients (Fig 1).8,10 It is a potent toxin with the ability to damage various parasites, fungi, bacteria, and mammalian cells.10,11 It is also able to stimulate histamine release from basophils and mast cells, and activate neutrophils.8,11 Given the evidence that eosinophils may be the major contributors to the pathophysiology of CRS, detection of eosinophilic infiltration and degranulation markers of eosinophils may be useful as a diagnostic tool. Since EDN and ECP are not specific for eosinophils and are also found in neutrophils, they are not optimal for diagnostic use.9 EPO is also not practical for diagnostic purposes due to its high degree of homology with other human peroxidases.9 eMBP, however, has a high degree of specificity for eosinophil granulocytes, making it the obvious choice among the four eosinophil granule proteins to be used as a candidate marker for diagnosing polypoid CRS.9-11
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The aim of this study was to detect and measure eMBP in the mucus of patients with polypoid CRS in Central Europe to use as a means to differentiate CRS from similarly presenting diseases and healthy controls.
Methods Patient Selection The Institutional Review Board of the University of Graz approved this study. All 44 patients (28 male, 16 female; mean age 44 years, range 18-73 years) provided informed consent and underwent a detailed history and physical examination, which included an allergy evaluation, skin prick testing, and radioimmunosorbent test/radioallergosorbent test). Patients were recruited regardless of current medications or preoperative treatment with systemic or topical steroids. Thirty-seven patients underwent endoscopic sinus surgery for different reasons, and seven were treated for reasons other than sinus disease (Table 1). All but three (three sinus healthy participants of control group 3) had imaging (computed tomography [CT] and/or magnetic resonance imaging) of their sinuses. The radiologic pathology of the paranasal sinuses was assessed using the LundMackay CT score.12
Patient Group Twenty-three patients undergoing functional endoscopic sinus surgery (FESS) who met criteria for polypoid CRS
Figure 1 (A) Representative chronic rhinosinusitis histologic specimen with an eosinophil cluster (yellow arrow) in mucus, and airway remodeling including basement membrane thickening (white arrow) and damaged epithelium (black arrow). (Hematoxylin and eosin; original magnification: ⫻630.) (B) A serial section of (A) showing eosinophil major basic protein (eMBP) within the intact eosinophils. In contrast, diffuse release of MBP is seen in eosinophil clusters in mucus. (Anti-eMBP; original magnification: ⫻630.)
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Table 1 Demographics of CRS patients and controls (n ⴝ 44 total)
Female Male Age (mean yrs) Diagnoses (n)
CRS patient group
Control group 1
Control group 2
Control group 3
10 13 44 Polypoid CRS (23)
2 4 33 CRS-like symptoms (6)
11 23
2 6
2 6 47 Pyocele (3) Fungus ball (2) Inverted papilloma (2) Antrochoanal polyp (1) 2 8
2 5 50 Chronic tonsillitis (3) Adenomatous parotid gland (2) Otosclerosis (2) 2 7
Allergy testing ⫹ Number (total) CRS, chronic rhinosinusitis.
served as our study group (10 female, 13 male; mean age 44 years, range 19-67 years).3 All symptoms of CRS were present for at least three months and all patients had characteristic CT findings, with a Lund-Mackay CT score of at least 13 points (mean score 19.0 points, range 13-24 points) in addition to the presence of polypoid changes in the middle meatus as seen on nasal endoscopy (Fig 2A). Preoperatively, seven patients did not receive either topical or systemic steroids, eight received topical steroids, and another eight received a systemic steroid boost within four weeks before surgery.
Control Group 1 Included were six patients with persisting CRS-like symptoms with only minimal mucosal thickening and mucus
retention on sinus CT (mean Lund-Mackay score 5.3 points, range 4-6 points) (Fig 2B). Nasal endoscopy findings were not indicative of polypoid CRS (4 male, 2 female; mean age 33 years, range 18-60 years). One patient was treated with topical steroids preoperatively.
Control Group 2 Included were eight patients (6 male, 2 female; mean age 47 years, range 18-67 years) who underwent FESS for reasons other than CRS: pyocele of isolated sinuses (n ⫽ 3), fungus ball (n ⫽ 2), inverted papilloma (n ⫽ 2), and antrochoanal polyp (n ⫽ 1). All patients of this group had sinus imaging indicative of disease in isolated sinuses (mean Lund-Mackay score 5.5 points, range 3-7 points) (Fig 2C). None of the patients in this group were treated with preoperative steroids.
Figure 2 Characteristic findings on computed tomography and endoscopy of chronic rhinosinusitis (CRS) patients with polyps (A) showing disseminated inflammatory mucosal thickening throughout the sinuses and polyps in the middle meatus. (B) Patients presenting with CRS-like symptoms but with only minimal isolated mucosal thickening (control group 1). (C) A patient with an antrochoanal polyp unrelated to CRS (control group 2).
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Released intranasal eosinophilic major basic protein . . .
Control Group 3 Included were seven patients (5 male, 2 female; mean age 50 years, range 20-73 years) with no CRS-symptoms who were treated for reasons other than sinus disease: chronic tonsillitis (n ⫽ 3), adenomatous parotid gland (n ⫽ 2), and otosclerosis (n ⫽ 2). In the four of seven who did undergo imaging studies, all showed clear sinuses (mean LundMackay score 1 point, range 0-2 points).
Mucus Collection In order to analyze the amount of eMBP in mucin, an appropriate amount of mucus (minimum 0.2 mL) had to be harvested from each patient. We used a special mucus collection device (Sinus Secretion Collector, MedtronicXomed Inc., Jacksonville, FL) without doing any preoperative decongestion or irrigation. The mucus was collected under endoscopic guidance from the nasal cavities, primarily from the middle meatus bilaterally. The mass of each mucus specimen was weighed, and a threefold excess of normal saline (0.15 M NaCl) was added. After vortexing for 10 seconds (⫻3), the mucus suspension was centrifuged, and the resulting supernatant fluid was frozen at ⫺70°C. Because we did not use any lysis buffer, which would be necessary to extract intracellular eMBP, only released “free” eMBP in the mucus was analyzed.
Quantitative Analysis of eMBP in Mucus An enzyme-linked immunosorbent assay using a monoclonal antibody specific for eMBP, designed and manufactured by Immco Diagnostics, Inc. (Buffalo, NY), was used to measure the eMBP levels. Samples were designated as positive or negative for eMBP using the lowest detection limit of 10 ng/mL.
Statistical Analysis Data collection was performed with Window’s Office Excel 2003 (Microsoft Corporation, Redmond, WA). Statistical analyses were done with SPSS Vs 14.0 (SPSS GmbH Software, Munich, Germany). 2 analyses were used for categorical variables (negative or positive with a sensitivity level of the assay of 10 ng/mL), and a P value of less than 0.05 was considered statistically significant. In addition, an analysis of variance (ANOVA) was performed to compare the polypoid CRS group with the three control groups.
Results Patient Group Twenty-three patients with clear criteria of polypoid CRS served as our patient group. Seven patients of this group had not been treated with steroids within four weeks prior to surgery, eight were on nasal steroids, and another eight had received a systemic steroid boost within four weeks before surgery. Eighty-seven percent were positive for eMBP. Of the three patients who were negative for eMBP, two were
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treated with systemic steroids. The mean concentration of detectable eMBP was 548.3 ng/mL (median 357.0, range 115-3600 ng/mL). Eleven of 23 patients (47.83%) had positive allergy testing to various allergens (Table 1).
Control Group 1 Six patients with persisting CRS-like symptoms, but with only minimal pathology on sinus CT, were included as control group 1. Only one patient used nasal steroids, and two had positive allergy testing (33.33%) (Table 1). No other patient received preoperative steroid treatment. None of these patients were eMBP positive.
Control Group 2 Eight patients that underwent FESS for reasons other than CRS served as control group 2: pyocele of isolated sinuses (n ⫽ 3), fungus ball (n ⫽ 2), inverted papilloma (n ⫽ 2), and antrochoanal polyp (n ⫽ 1). None of the patients in this group were treated with steroids preoperatively, and two patients (25%) had a confirmed allergy (Table 1). Only one patient was eMBP positive. This patient was clinically and radiologically suspected of having a fungus ball in one maxillary sinus. However, intraoperatively and histologically, the diagnosis was not proven and was changed to sinonasal polyps.
Control Group 3 Seven patients with no symptoms of CRS who were treated for reasons other than sinus disease were included: chronic tonsillitis (n ⫽ 3), adenomatous parotid gland (n ⫽ 2), and otosclerosis (n ⫽ 2). Two patients (28.57%) had positive allergy tests (Table 1). None of these patients was eMBP positive.
Group Comparison All findings comparing the CRS patient group to each of the three control groups were statistically significant (P ⬍ 0.001) (Fig 3) on both the 2 analyses and the ANOVA analyses.
Discussion The importance of eosinophilic inflammation in the pathogenesis of CRS and an association between eosinophilic infiltration, degranulation, and the epithelial damage in CRS has long been postulated.4,5,13 Because of the well-established toxicity of eosinophil granule proteins to epithelial surfaces, especially eMBP, a link between granule deposition and the epithelial damage in CRS is likely given its newfound high concentrations in CRS patients.10,13,14 This is the first study outside of the United States that was able to detect eMBP in the nasal mucus of patients with polypoid CRS, a finding that supports previous observations and suggests a similar, if not identical, disease process driving polypoid CRS in Europe.10 We found significantly increased eMBP levels in 87 percent of patients with pol-
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Figure 3 Eighty-seven percent of chronic rhinosinusitis (CRS) patients and only one patient in control group 2 had detectable levels of eosinophil major basic protein (eMBP). Remaining patients were eMBP-negative (P ⬍ 0.001 between patient group and each of the control groups). Control group 1, CRS symptoms without computed tomography signs; control group II, non-CRS sinus pathology; control group III, no sinus pathology.
ypoid CRS. These findings again suggest an important role of eMBP and might further aid to reveal the underlying pathogenesis and pathophysiology of CRS. A few remaining issues require further consideration. Although this study was able to show a clear relationship between patients with polypoid CRS and elevated levels of eMBP, this study is limited by its small sample size and lack of standardization regarding preoperative treatment regimens. These shortcomings do not allow us to speak further about the absolute values of eMBP measured herein, since steroids reduce the number of eosinophils and ultimately the amount of detectable eMBP.15,16 Additionally, it has been debated whether eosinophilic infiltration in patients with CRS is independent or codependent of an immunoglobulin E-mediated type 1 allergy.7 Therefore, allergy testing was performed. Although patients with polypoid CRS were more likely to have positive allergy testing compared to controls (patient group: 47.83%; control group 1: 33.33%; control group 2: 25%; control group 3: 28.57%), this difference did not show statistical significance (P ⬎ 0.6). Thus, this suggests that polypoid CRS is not necessarily associated with an atopic disposition. From another standpoint, allergic patients in the control groups were negative for eMBP in the mucus, suggesting that eMBP is not released in allergic rhinitis. This may indicate a different degranulation pattern between polypoid CRS and allergic rhinitis. Currently, no immunologic-based test exists for the diagnosis of CRS. In this small series, measuring intranasal eMBP was able to identify 87 percent (sensitivity) of patients with polypoid CRS as confirmed by CT and endoscopy, and was 100 percent specific. It must be emphasized
that in this study, we focused on patients with polypoid CRS; further trials are necessary to examine whether the results and conclusions can be expanded to CRS without polypoid mucosa. Further development of an assay used to detect eMBP in nasal mucus may become a simple and standardized diagnostic tool able to divide those patients with actual CRS disease from those who present with CRSlike symptoms yet lack objective criteria, similar to our study. Specifically, the fact that eMBP was not detected in allergic rhinitis patients, which confirms previous studies, might yield a diagnostic test differentiating CRS from allergic rhinitis.17 Such a tool combined with a simplified collection method (e.g., swab) would be most beneficial to the general practitioner who lacks other means to screen for the differential diagnosis of CRS (CT, endoscopy), and as a referral criterion to send patients to specialists. Furthermore, the application may be wider reaching than for just CRS patients. For example, eMBP also plays an important role in the pathophysiology of asthma.14,18 It is deposited at sites of respiratory epithelial damage and is capable of inducing hyperreactivity of bronchial smooth muscle and activating complement.18-20 At present, there is a movement to improve prevention and early intervention methods through better diagnosis among all diseases. With the application of this method, we may be able to join in this effort for polypoid CRS.
Author Information From the Department of Otorhinolaryngology–Head and Neck Surgery, Medical University of Graz (Drs. Schmid, Habermann, Braun, Gugatschka,
Schmid et al
Released intranasal eosinophilic major basic protein . . .
and Stammberger), Graz, Austria; and the Department of Otolaryngology, University at Buffalo, The State University of New York (Mr. Oriel and Ms. Smietana), Buffalo, NY. Corresponding author: Christoph Schmid, MD, Department of Otorhinolaryngology–Head and Neck Surgery, Medical University of Graz, Auenbruggerplatz 26-28, Graz 8036, Austria. E-mail address: [email protected].
Author Contributions Christoph Schmid, study design, acquisition of probands, statistical analysis, interpretation of data, approval of article; Walter Habermann, statistical analysis, acquisition of probands; Hannes Braun, acquisition of probands; Markus Gugatschka, statistical analysis, acquisition of probands; Brad S. Oriel, statistical analysis, interpretation of data, approval of article; Janel A. Smietana, statistical analysis, interpretation of data, approval of article; Heinz Stammberger, study design, interpretation of data, approval of article.
Disclosures Competing interests: None. Sponsorships: None.
References 1. Pleis JR, Lethbridge-Çejku M. Summary health statistics for U.S. adults: National Health Interview Survey, 2006. Vital Health Stat 2007;10:1–153. 2. Deutsche Gesellschaft für Hals-Nasen-Ohren-Heilkunde, Kopf- und HalsChirurgie. AWMF Reg.-Nr. 017/049. Leitlinie Rhinosinusitis. Available at: http://www.uni-duesseldorf.de/awmf/ll/017-049.htm. Accessed September 10, 2009. 3. Fokkens W, Lund V, Mullol J. European position paper on rhinosinusitis and nasal polyps. Rhinol Suppl 2007;20:1–136. 4. Bachert C, Gevaert P, van Cauwenberge P. Staphylococcus aureus superantigens and airway disease. Curr Allergy 2002;2:252– 8. 5. Ponikau JU, Sherris DA, Kita H. The role of ubiquitous airborne fungi in chronic rhinosinusitis. Clin Allergy Immunol 2007;20: 177– 84.
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6. Braun H, Buzina W, Freudenschuss K, et al. Eosinophilic fungal rhinosinusitis: a common disorder in Europe? Laryngoscope 2003; 113:264 –9. 7. Hamilos DL, Leung DY, Wood R, et al. Evidence for distinct cytokine expression in allergic versus nonallergic chronic sinusitis. J Allergy Clin Immunol 1995;96:537– 44. 8. Rothenberg ME, Hogan SP. The eosinophil. Annu Rev Immunol 2006;24:147–74. 9. Abu-Ghazaleh RI, Dunnette SL, Loegering DA, et al. Eosinophil granule proteins in peripheral blood granulocytes. J Leukoc Biol 1992; 52:611– 8. 10. Ponikau JU, Sherris DA, Kephart GM, et al. Striking deposition of toxic eosinophil major basic protein in mucus: implications for chronic rhinosinusitis. J Allergy Clin Immunol 2005;116:362–9. 11. Motojima S, Frigas E, Loegering DA, et al. Toxicity of eosinophil cationic proteins for guinea pig tracheal epithelium in vitro. Am Rev Respir Dis 1989;139:801–5. 12. Lund VJ, Mackay IS. Staging in rhinosinusitus. Rhinology 1993;31: 183– 4. 13. Harlin SL, Ansel DG, Lane SR, et al. A clinical and pathologic study of chronic sinusitis: the role of the eosinophil. J Allergy Clin Immunol 1988;81:867–75. 14. Gleich GJ, Loegering DA, Frigas E, et al. The eosinophil granule major basic protein: biological activities and relationship to bronchial asthma. Monogr Allergy 1983;18:277– 83. 15. Watanabe K, Shirasaki H, Kanaizumi E, et al. Effects of glucocorticoids on infiltrating cells and epithelial cells of nasal polyps. Ann Otol Rhinol Laryngol 2004;113:465–73. 16. Burgel PR, Cardell LO, Ueki IF, et al. Intranasal steroids decrease eosinophils but not mucin expression in nasal polyps. Eur Respir J 2004;24:594 – 600. 17. Kita H, Jorgensen RK, Reed CE, et al. Mechanism of topical glucocorticoid treatment of hay fever: IL-5 and eosinophil activation during natural allergen exposure are suppressed, but IL-4, IL-6, and IgE antibody production are unaffected. J Allergy Clin Immunol 2000;106:521–9. 18. Frigas E, Gleich GJ. The eosinophil and the pathophysiology of asthma. J Allergy Clin Immunol 1986;77:527–37. 19. Gundel RH, Letts LG, Gleich GJ. Human eosinophil major basic protein induces airway constriction and airway hyperresponsiveness in primates. J Clin Invest 1991;87:1470 –3. 20. Weiler JM, Gleich GJ. Eosinophil granule major basic protein regulates generation of classical and alternative-amplification pathway C3 convertases in vitro. J Immunol 1988;140:1605–10.
ORIGINAL RESEARCH–ALLERGY
Released intranasal eosinophilic major basic protein as a diagnostic marker for polypoid chronic rhinosinusitis Christoph Schmid, MD, Walter Habermann, MD, Hannes Braun, MD, Markus Gugatschka, MD, Brad S. Oriel, Janel A. Smietana, and Heinz Stammberger, MD, Graz, Austria; and Buffalo, NY No sponsorships or competing interests have been disclosed for this article. ABSTRACT OBJECTIVE: To investigate the hypothesis that eosinophil major basic protein is released in high concentrations in the nasal mucus of patients with polypoid chronic rhinosinusitis. STUDY DESIGN: Single center, open, prospective trial. SETTING: Medical University of Graz, Austria. SUBJECTS AND METHODS: Patients with polypoid chronic rhinosinusitis (n ⫽ 23) were compared to three different control groups: patients with chronic rhinosinusitis-like symptoms but without general mucosal thickening, patients who underwent functional endoscopic sinus surgery for reasons other than chronic rhinosinusitis, and patients without sinus disease (total n ⫽ 21). Mucus was harvested from each patient using a standardized technique and analyzed for eosinophil major basic protein with an enzyme-linked immunosorbent assay. RESULTS: In the patient group with polypoid chronic rhinosinusitis, 20 of 23 (87%) were positive for eosinophil major basic protein. In contrast, only one control patient was positive, whereas the remainder had no detectable amount of eosinophil major basic protein in the mucus (P ⬍ 0.001 vs chronic rhinosinusitis). CONCLUSION: Toxic eosinophil major basic protein levels are elevated in polypoid chronic rhinosinusitis patients compared to control groups that have similar clinical presentations but upon closer examination turn out not to have chronic rhinosinusitis. In the future, the detection of eosinophil major basic protein in nasal mucus may become a sensitive and specific marker for chronic rhinosinusitis and a helpful diagnostic tool. © 2010 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved.
C
hronic sinusitis afflicts many individuals worldwide. The 2006 National Health Interview Survey for the civilian noninstitutionalized adult population of the United States found the prevalence of chronic sinusitis in the adult population to be 13.8 percent, making it one of the most
common chronic diseases.1 Additionally, the German Society for Otorhinolaryngology–Head and Neck Surgery reported that the diagnosis of chronic sinusitis was made 2.6 million times in Germany in 2002, which resulted in 3.4 million medical prescriptions.2 Currently, controversy exists over the etiology and pathogenesis of chronic rhinosinusitis (CRS).3 Two hypothesized immunologic mechanisms are discussed below. Some authors propose that Staphylococcus aureus enterotoxins are implicated in the pathophysiology of CRS. These enterotoxins function as superantigens, resulting in local production of polyclonal immunoglobulin E, which may contribute to the severe eosinophilic inflammation mediated primarily by mast cells.4 According to the second hypothesis, most CRS is caused by a pathologic immune response to airborne fungi.5 These fungi are ubiquitous and can be detected in the nasal mucus of more than 90 percent of CRS patients as well as healthy subjects.5,6 However, only CRS patients produce interleukin (IL)-5 and IL-13 in response to the fungi, which are crucial cytokines known to drive the eosinophilic inflammation in CRS.7 The eosinophils do not remain in the tissue but actively move to the mucus, where they form clusters around the fungi.5 Regardless of the immunological mechanism proposed, both lead to an eosinophilic-dominated inflammation. Thus, the postulated damage of the tissue by toxic granular proteins of eosinophilic granulocytes may be valid for both theories. Eosinophilic granulocytes contain four principal cytotoxic proteins in their granules. The granular matrix is composed of eosinophil peroxidase (EPO), eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). The characteristic granule core consists of eosinophil major basic protein (eMBP). EPO constitutes about 25 percent of the total protein mass of eosinophil granules.8,9 In the presence of hydrogen peroxide, EPO functions as a peroxidase with strong helminthotoxic activities, but it is also able to damage respiratory epithelium. EDN has mul-
Received September 16, 2009; revised May 11, 2010; accepted May 14, 2010.
0194-5998/$36.00 © 2010 American Academy of Otolaryngology–Head and Neck Surgery Foundation. All rights reserved. doi:10.1016/j.otohns.2010.05.017
Schmid et al
Released intranasal eosinophilic major basic protein . . .
tiple functions. It is a powerful neurotoxin, a potent ribonuclease, and possesses weak helminthotoxic activities.8,9 ECP, like EDN, is a member of the ribonuclease gene superfamily. Although ECP is toxic to bacteria, helminthes, protozoa, and mammalian cells, it also has a number of additional noncytotoxic effects, including suppression of T-cell responses, induction of mast cell degranulation, and stimulation of airway mucus secretion.8,9 eMBP is the principle protein in the eosinophil granule, comprising roughly 50 percent of the total granule protein mass, and has been found at concentrations exceeding more than a thousand times the amount needed to damage epithelium in the nasal mucus of CRS patients (Fig 1).8,10 It is a potent toxin with the ability to damage various parasites, fungi, bacteria, and mammalian cells.10,11 It is also able to stimulate histamine release from basophils and mast cells, and activate neutrophils.8,11 Given the evidence that eosinophils may be the major contributors to the pathophysiology of CRS, detection of eosinophilic infiltration and degranulation markers of eosinophils may be useful as a diagnostic tool. Since EDN and ECP are not specific for eosinophils and are also found in neutrophils, they are not optimal for diagnostic use.9 EPO is also not practical for diagnostic purposes due to its high degree of homology with other human peroxidases.9 eMBP, however, has a high degree of specificity for eosinophil granulocytes, making it the obvious choice among the four eosinophil granule proteins to be used as a candidate marker for diagnosing polypoid CRS.9-11
387
The aim of this study was to detect and measure eMBP in the mucus of patients with polypoid CRS in Central Europe to use as a means to differentiate CRS from similarly presenting diseases and healthy controls.
Methods Patient Selection The Institutional Review Board of the University of Graz approved this study. All 44 patients (28 male, 16 female; mean age 44 years, range 18-73 years) provided informed consent and underwent a detailed history and physical examination, which included an allergy evaluation, skin prick testing, and radioimmunosorbent test/radioallergosorbent test). Patients were recruited regardless of current medications or preoperative treatment with systemic or topical steroids. Thirty-seven patients underwent endoscopic sinus surgery for different reasons, and seven were treated for reasons other than sinus disease (Table 1). All but three (three sinus healthy participants of control group 3) had imaging (computed tomography [CT] and/or magnetic resonance imaging) of their sinuses. The radiologic pathology of the paranasal sinuses was assessed using the LundMackay CT score.12
Patient Group Twenty-three patients undergoing functional endoscopic sinus surgery (FESS) who met criteria for polypoid CRS
Figure 1 (A) Representative chronic rhinosinusitis histologic specimen with an eosinophil cluster (yellow arrow) in mucus, and airway remodeling including basement membrane thickening (white arrow) and damaged epithelium (black arrow). (Hematoxylin and eosin; original magnification: ⫻630.) (B) A serial section of (A) showing eosinophil major basic protein (eMBP) within the intact eosinophils. In contrast, diffuse release of MBP is seen in eosinophil clusters in mucus. (Anti-eMBP; original magnification: ⫻630.)
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Table 1 Demographics of CRS patients and controls (n ⴝ 44 total)
Female Male Age (mean yrs) Diagnoses (n)
CRS patient group
Control group 1
Control group 2
Control group 3
10 13 44 Polypoid CRS (23)
2 4 33 CRS-like symptoms (6)
11 23
2 6
2 6 47 Pyocele (3) Fungus ball (2) Inverted papilloma (2) Antrochoanal polyp (1) 2 8
2 5 50 Chronic tonsillitis (3) Adenomatous parotid gland (2) Otosclerosis (2) 2 7
Allergy testing ⫹ Number (total) CRS, chronic rhinosinusitis.
served as our study group (10 female, 13 male; mean age 44 years, range 19-67 years).3 All symptoms of CRS were present for at least three months and all patients had characteristic CT findings, with a Lund-Mackay CT score of at least 13 points (mean score 19.0 points, range 13-24 points) in addition to the presence of polypoid changes in the middle meatus as seen on nasal endoscopy (Fig 2A). Preoperatively, seven patients did not receive either topical or systemic steroids, eight received topical steroids, and another eight received a systemic steroid boost within four weeks before surgery.
Control Group 1 Included were six patients with persisting CRS-like symptoms with only minimal mucosal thickening and mucus
retention on sinus CT (mean Lund-Mackay score 5.3 points, range 4-6 points) (Fig 2B). Nasal endoscopy findings were not indicative of polypoid CRS (4 male, 2 female; mean age 33 years, range 18-60 years). One patient was treated with topical steroids preoperatively.
Control Group 2 Included were eight patients (6 male, 2 female; mean age 47 years, range 18-67 years) who underwent FESS for reasons other than CRS: pyocele of isolated sinuses (n ⫽ 3), fungus ball (n ⫽ 2), inverted papilloma (n ⫽ 2), and antrochoanal polyp (n ⫽ 1). All patients of this group had sinus imaging indicative of disease in isolated sinuses (mean Lund-Mackay score 5.5 points, range 3-7 points) (Fig 2C). None of the patients in this group were treated with preoperative steroids.
Figure 2 Characteristic findings on computed tomography and endoscopy of chronic rhinosinusitis (CRS) patients with polyps (A) showing disseminated inflammatory mucosal thickening throughout the sinuses and polyps in the middle meatus. (B) Patients presenting with CRS-like symptoms but with only minimal isolated mucosal thickening (control group 1). (C) A patient with an antrochoanal polyp unrelated to CRS (control group 2).
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Released intranasal eosinophilic major basic protein . . .
Control Group 3 Included were seven patients (5 male, 2 female; mean age 50 years, range 20-73 years) with no CRS-symptoms who were treated for reasons other than sinus disease: chronic tonsillitis (n ⫽ 3), adenomatous parotid gland (n ⫽ 2), and otosclerosis (n ⫽ 2). In the four of seven who did undergo imaging studies, all showed clear sinuses (mean LundMackay score 1 point, range 0-2 points).
Mucus Collection In order to analyze the amount of eMBP in mucin, an appropriate amount of mucus (minimum 0.2 mL) had to be harvested from each patient. We used a special mucus collection device (Sinus Secretion Collector, MedtronicXomed Inc., Jacksonville, FL) without doing any preoperative decongestion or irrigation. The mucus was collected under endoscopic guidance from the nasal cavities, primarily from the middle meatus bilaterally. The mass of each mucus specimen was weighed, and a threefold excess of normal saline (0.15 M NaCl) was added. After vortexing for 10 seconds (⫻3), the mucus suspension was centrifuged, and the resulting supernatant fluid was frozen at ⫺70°C. Because we did not use any lysis buffer, which would be necessary to extract intracellular eMBP, only released “free” eMBP in the mucus was analyzed.
Quantitative Analysis of eMBP in Mucus An enzyme-linked immunosorbent assay using a monoclonal antibody specific for eMBP, designed and manufactured by Immco Diagnostics, Inc. (Buffalo, NY), was used to measure the eMBP levels. Samples were designated as positive or negative for eMBP using the lowest detection limit of 10 ng/mL.
Statistical Analysis Data collection was performed with Window’s Office Excel 2003 (Microsoft Corporation, Redmond, WA). Statistical analyses were done with SPSS Vs 14.0 (SPSS GmbH Software, Munich, Germany). 2 analyses were used for categorical variables (negative or positive with a sensitivity level of the assay of 10 ng/mL), and a P value of less than 0.05 was considered statistically significant. In addition, an analysis of variance (ANOVA) was performed to compare the polypoid CRS group with the three control groups.
Results Patient Group Twenty-three patients with clear criteria of polypoid CRS served as our patient group. Seven patients of this group had not been treated with steroids within four weeks prior to surgery, eight were on nasal steroids, and another eight had received a systemic steroid boost within four weeks before surgery. Eighty-seven percent were positive for eMBP. Of the three patients who were negative for eMBP, two were
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treated with systemic steroids. The mean concentration of detectable eMBP was 548.3 ng/mL (median 357.0, range 115-3600 ng/mL). Eleven of 23 patients (47.83%) had positive allergy testing to various allergens (Table 1).
Control Group 1 Six patients with persisting CRS-like symptoms, but with only minimal pathology on sinus CT, were included as control group 1. Only one patient used nasal steroids, and two had positive allergy testing (33.33%) (Table 1). No other patient received preoperative steroid treatment. None of these patients were eMBP positive.
Control Group 2 Eight patients that underwent FESS for reasons other than CRS served as control group 2: pyocele of isolated sinuses (n ⫽ 3), fungus ball (n ⫽ 2), inverted papilloma (n ⫽ 2), and antrochoanal polyp (n ⫽ 1). None of the patients in this group were treated with steroids preoperatively, and two patients (25%) had a confirmed allergy (Table 1). Only one patient was eMBP positive. This patient was clinically and radiologically suspected of having a fungus ball in one maxillary sinus. However, intraoperatively and histologically, the diagnosis was not proven and was changed to sinonasal polyps.
Control Group 3 Seven patients with no symptoms of CRS who were treated for reasons other than sinus disease were included: chronic tonsillitis (n ⫽ 3), adenomatous parotid gland (n ⫽ 2), and otosclerosis (n ⫽ 2). Two patients (28.57%) had positive allergy tests (Table 1). None of these patients was eMBP positive.
Group Comparison All findings comparing the CRS patient group to each of the three control groups were statistically significant (P ⬍ 0.001) (Fig 3) on both the 2 analyses and the ANOVA analyses.
Discussion The importance of eosinophilic inflammation in the pathogenesis of CRS and an association between eosinophilic infiltration, degranulation, and the epithelial damage in CRS has long been postulated.4,5,13 Because of the well-established toxicity of eosinophil granule proteins to epithelial surfaces, especially eMBP, a link between granule deposition and the epithelial damage in CRS is likely given its newfound high concentrations in CRS patients.10,13,14 This is the first study outside of the United States that was able to detect eMBP in the nasal mucus of patients with polypoid CRS, a finding that supports previous observations and suggests a similar, if not identical, disease process driving polypoid CRS in Europe.10 We found significantly increased eMBP levels in 87 percent of patients with pol-
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Otolaryngology–Head and Neck Surgery, Vol 143, No 3, September 2010
Figure 3 Eighty-seven percent of chronic rhinosinusitis (CRS) patients and only one patient in control group 2 had detectable levels of eosinophil major basic protein (eMBP). Remaining patients were eMBP-negative (P ⬍ 0.001 between patient group and each of the control groups). Control group 1, CRS symptoms without computed tomography signs; control group II, non-CRS sinus pathology; control group III, no sinus pathology.
ypoid CRS. These findings again suggest an important role of eMBP and might further aid to reveal the underlying pathogenesis and pathophysiology of CRS. A few remaining issues require further consideration. Although this study was able to show a clear relationship between patients with polypoid CRS and elevated levels of eMBP, this study is limited by its small sample size and lack of standardization regarding preoperative treatment regimens. These shortcomings do not allow us to speak further about the absolute values of eMBP measured herein, since steroids reduce the number of eosinophils and ultimately the amount of detectable eMBP.15,16 Additionally, it has been debated whether eosinophilic infiltration in patients with CRS is independent or codependent of an immunoglobulin E-mediated type 1 allergy.7 Therefore, allergy testing was performed. Although patients with polypoid CRS were more likely to have positive allergy testing compared to controls (patient group: 47.83%; control group 1: 33.33%; control group 2: 25%; control group 3: 28.57%), this difference did not show statistical significance (P ⬎ 0.6). Thus, this suggests that polypoid CRS is not necessarily associated with an atopic disposition. From another standpoint, allergic patients in the control groups were negative for eMBP in the mucus, suggesting that eMBP is not released in allergic rhinitis. This may indicate a different degranulation pattern between polypoid CRS and allergic rhinitis. Currently, no immunologic-based test exists for the diagnosis of CRS. In this small series, measuring intranasal eMBP was able to identify 87 percent (sensitivity) of patients with polypoid CRS as confirmed by CT and endoscopy, and was 100 percent specific. It must be emphasized
that in this study, we focused on patients with polypoid CRS; further trials are necessary to examine whether the results and conclusions can be expanded to CRS without polypoid mucosa. Further development of an assay used to detect eMBP in nasal mucus may become a simple and standardized diagnostic tool able to divide those patients with actual CRS disease from those who present with CRSlike symptoms yet lack objective criteria, similar to our study. Specifically, the fact that eMBP was not detected in allergic rhinitis patients, which confirms previous studies, might yield a diagnostic test differentiating CRS from allergic rhinitis.17 Such a tool combined with a simplified collection method (e.g., swab) would be most beneficial to the general practitioner who lacks other means to screen for the differential diagnosis of CRS (CT, endoscopy), and as a referral criterion to send patients to specialists. Furthermore, the application may be wider reaching than for just CRS patients. For example, eMBP also plays an important role in the pathophysiology of asthma.14,18 It is deposited at sites of respiratory epithelial damage and is capable of inducing hyperreactivity of bronchial smooth muscle and activating complement.18-20 At present, there is a movement to improve prevention and early intervention methods through better diagnosis among all diseases. With the application of this method, we may be able to join in this effort for polypoid CRS.
Author Information From the Department of Otorhinolaryngology–Head and Neck Surgery, Medical University of Graz (Drs. Schmid, Habermann, Braun, Gugatschka,
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Released intranasal eosinophilic major basic protein . . .
and Stammberger), Graz, Austria; and the Department of Otolaryngology, University at Buffalo, The State University of New York (Mr. Oriel and Ms. Smietana), Buffalo, NY. Corresponding author: Christoph Schmid, MD, Department of Otorhinolaryngology–Head and Neck Surgery, Medical University of Graz, Auenbruggerplatz 26-28, Graz 8036, Austria. E-mail address: [email protected].
Author Contributions Christoph Schmid, study design, acquisition of probands, statistical analysis, interpretation of data, approval of article; Walter Habermann, statistical analysis, acquisition of probands; Hannes Braun, acquisition of probands; Markus Gugatschka, statistical analysis, acquisition of probands; Brad S. Oriel, statistical analysis, interpretation of data, approval of article; Janel A. Smietana, statistical analysis, interpretation of data, approval of article; Heinz Stammberger, study design, interpretation of data, approval of article.
Disclosures Competing interests: None. Sponsorships: None.
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