Lysosomal exoglycosidases in nasal polyps

otolaryngologia polska 67 (2013) 192–197

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Original research article/Artykuł oryginalny

Lysosomal exoglycosidases in nasal polyps Sylwia Chojnowska 1,*, Alina Minarowska 2, Małgorzata Knaś 3, Anna Niemcunowicz-Janica 4, Paweł Kołodziejczyk 5, Beata Zalewska-Szajda 6, Alina Kępka 7, Łukasz Minarowski 8, Napoleon Waszkiewicz 9, Krzysztof Zwierz 5, Sławomir Dariusz Szajda 9 1

Medical Institute, College of Computer Science and Business Administration, Lomza, Poland Department of Anatomy, Poland 3 Research Laboratory of Cosmetology, Poland 4 Department of Forensic Medicine, Poland 5 Medical College of the Universal Education Society, Lomza, Poland 6 Department of Radiology, Medical University Children Hospital, Bialystok, Poland 7 The Children's Memorial Health Institute, Warsaw, Poland 8 Department of Lung Diseases and Tuberculosis, Medical University of Bialystok, Poland 9 Department of the Emergency Medicine and Disasters, Medical University of Bialystok, Poland 2

article info

abstract

Article history:

Introduction: Nasal polyps are smooth outgrowths assuming a shape of grapes, formed

Received: 14.05.2013

from the nasal mucosa, limiting air flow by projecting into a lumen of a nasal cavity. Up

Accepted: 22.05.2013

to now the surgical resection is the best method of their treatment, but etiology and

Available online: 24.05.2013

pathogenesis of the nasal polyps is not yet fully established. Aim of the study: The aim of the study was the assessment of the selected lysosomal exoglycosidases activity in

Keywords:  Lysosomal exoglycosidases  a-Fucosidase

the nasal polyps. In this study the activity of b-galactosidase, a-mannosidase and a-

 b-Galactosidase  a-Mannosidase

during mucotomy from 20 patients (10 F, 10 M). Results: We observed significant lower

 Nasal polyps

polyps (P) in comparison to control healthy nasal mucosa (C). In nasal polyp tissue (P) no

fucosidase was determined in the tissue of the nasal polyps obtained from 40 patients (10 F, 30 M) and control tissues derived from mucosa of lower nasal conchas obtained values of GAL, FUC and tendency to decrease of MAN and GLU concentration in nasal differences of GAL, MAN and FUC specific activity in comparison to control mucosa (C) were found. Conclusions: Our research supports bioelectrical theory of the nasal polyps pathogenesis and directs attention at research on glycoconjugates and glycosidases of the nasal mucosa extracellular matrix. © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

* Corresponding author at: Medical Institute, College of Computer Science and Business Administration, Akademicka Str. 14, 18-400 Łomża, Poland. Tel.: +48 86 215 59 53; fax: +48 86 215 66 01. E-mail address: [email protected] (S. Chojnowska). 0030-6657/$ – see front matter © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

http://dx.doi.org/10.1016/j.otpol.2013.05.004

otolaryngologia polska 67 (2013) 192–197

Introduction Nasal polyps (gr. polýpous – polypod, i.e. many pads) [1] are smooth, grapes shape outgrowths of the nasal mucosa, projecting into nasal cavity, reducing nasal air flow, and creating danger of extra- and intra-cranial complications [2– 4]. Nasal polyps affect from 0.2–5% to 28% of the world population, mostly men in Europe, after 20 years of life with a mean age 38–39 years [5–9]. Nasal polyps accompanying by eosinophilia consist 80–90% of all nasal polyps, and the rest is accompanied by neutrophilia [10–12]. Local application of glucocorticosteroids is effective in relieving symptoms of the eosinophilic nasal polyps, decreasing their size and usually preventing against relapse, but neutrophilic polyps did not answer corticosteroid treatment [13, 14]. Recently new therapies are being tested for nasal polyps treatment: antiallergic drugs with monoclonal antibodies against IgE and antibiotics directed against bacterial biofilm [4, 15–19]. In the nasal polyps treatment, the best results obtained by polypectomy, particularly in large polyps and polyps occupying nasal sinuses [20, 21]. However in up to 87% of polypectomised patients recurrence of was observed [3, 18]. Majority of physicians believe that nasal polyps are final stage of a chronic inflammatory process in the nasal mucosal membrane [22–24]. In 1994 Bernstein proposed inflammatorybioelectric theory of nasal polyps formation based on abnormal chloride channels favoring increased absorption of sodium cations, changed composition of nasal mucus, increased water penetration to nasal mucosa intracellular matrix, swelling of mucosal tissue [9, 27]. The Bernstein theory is in agreement with reported higher (in comparison to healthy nasal mucosa) ability of the nasal polyps tissue to absorb Na+ [27, 28]. Recently attempts to correlate nasal polyps incidence with eosinophilia and eosinophilic inflammation of mucosa were reported [10, 11]. However, it was reported that only 5% of persons with nasal polyps suffer from allergy, with exception of fungal allergy (allergic fungal rhinosinusitis – AFRS) where frequency of nasal polyps exceeded 85% [2, 7, 25, 26]. It is generally accepted that in rhinosinuitis with nasal polyps formation following mechanisms are involved: excessive fibroblast proliferation after injury, degranulation of mastocytes, histamine release, eosinophiles infiltration and edema with exudation [7, 28]. It appears that, independently of etiology nasal polyps formation should be related to changes in structure, concentration and metabolism of substances building nasal mucosa and among them glycoconjugates (glycoproteins, glycolipids, and proteoglycans) constituting cell membranes, extracellular matrix and biofilm covering nasal mucosa [17, 19]. In catabolism of glycoconjugates saccharide chains lysosomal exoglycosidases are involved [37]. Thus, the aim of our work was an evaluation of selected lysosomal exoglycosidases in pathogenesis of the nasal polyps.

Materials and methods Patients. Nasal polyps were obtained during polypectomy from 40 patients (10 women and 30 men, aged 23–84 years)

193

with diagnosed chronic rhinosinusitis with nasal polyps. Control tissues were derived from mucosa of lower nasal conchas obtained during mucotomy operations from 20 patients (10 women and 10 men, aged 23–61 years). Biological material processing. To perform histopathological examination, fragments of polyps and normal nasal tissue were immersed in 10% formalin and subsequently stained with H + E. For biochemical investigations fragments of polyps and normal nasal mucosa were homogenized in homogenizer (Ultra-Turrax T8, Germany). Homogenates were centrifuged at 12 000  g for 20 min at 4 8C before further procedures. Glycosidases activity. Activity of exoglycosidases in supernatants of centrifuged homogenates was determined by Chatterjee et al. method [29] in modification of Zwierz et al. [30] and Marciniak et al. [31] with 4-nitrophenylderivatives of the appropriate sugars. Total protein concentration was determined by the Lowry et al. method [32]. Ethical issues. Informed written consent was obtained from all the participants after explanation of the nature, purpose, and potential risk of the study. The study was approved by the Bioethical Committee of the Medical University of Białystok, Poland (R-I-002/46/2007). Statistical analysis. All variables were checked for normality. Results were expressed as the mean  SD. P values less than 0.05 were considered significant. Statistical analysis was performed using packet Statistica 6.0 (StatSoft, Poland).

Results Normal nasal mucosa and nasal polyp tissue with pathological findings (submucosal edema, inflammatory infiltration, superficial hyperplasia) are presented in Fig. 1. We observed significant lower values of GAL, FUC and tendency to decrease MAN and GLU concentration in nasal polyps (P) in comparison to controls (C) (Fig. 2). In nasal polyp tissue (P) no differences of GAL, MAN and FUC specific activity in comparison to control mucosa were observed (C) (Fig. 3). It is worthy to note a similar decrease in concentration of activity for GAL, MAN, and FUC (from 0.71 for FUC to 0.78 for MAN) in polypous tissue in comparison to controls (Table I) and the same level of tendency to decrease in specific activity, i.e. from 0.79 to FUC to 0.92 for MAN (Table I).

Discussion In pathological situations connected with tissue remodeling such as colorectal cancer, pancreatic cancer, renal cancer, salivary gland tumors [36], alcoholism [37], Lyme disease [50] significant increase in lysosomal exoglycosidases activity was observed. Decrease in concentration of the activity of lysosomal exoglycosidases in nasal polyps tissue is contraindicatory to active inflammatory process in nasal polyps. In nasal polyps we observed significant decrease in concentration the activity of GAL, FUC, and tendency to

194

otolaryngologia polska 67 (2013) 192–197

[(Fig._1)TD$IG]

Fig. 1 – (A) Normal mucosa (H + E, magnification 20T). (B). Submucosal edema of polypous tissue (H + E, magnification 10T). (C) Polypous tissue with inflammation foci (H + E, magnification 10T). (D) Stratified squamous epithelium hyperplasia on the surface of a polyp (H + E, magnification 20T)

decrease MAN concentration (Table I, Fig. 2). In polypous tissue, in comparison to control nasal mucosa, specific activity of GAL, MAN, and FUC had tendency to decrease (Table II, Fig. 3). Decrease in concentration of GAL and FUC in association with tendency to decrease in concentration of MAN and all exoglycosidases specific activities is in agreement with accumulation of water in polypous tissue. Our

results are contradictory to existence of full symptomatic eosinophilic [11, 33] or neutrophilic [21, 33] inflammation in nasal polyps, as inflammatory state is connected with significant increase in activity of lysosomal exoglycosidases [37]. It is worthy to note that level of decrease in exoglycosidases concentration at nasal polyps tissue was similar for all exoglycosidases tested, e.g. from 0.71 for FUC to 0.78 for

[(Fig._2)TD$IG]

[(Fig._3)TD$IG]

Fig. 2 – Lysosomal exoglycosidases concentrations in nasal polyps and control tissues; c – control, p – polyp, *p < 0.05, **p < 0.01

Fig. 3 – Lysosomal exoglycosidases specific activities in nasal polyps and control tissues; c – control, p – polyp

otolaryngologia polska 67 (2013) 192–197

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Table I – Proportions of lysosomal exoglycosidases concentrations in nasal polyps and control tissues Exoglycosidase

Tissue

N

Concentration of activity (pKat/1 g tissue)

p

GAL

control polyp control polyp control polyp

20 40 20 40 20 40

259.1  93.3 195.7  89.9 194.7  71.5 138.6  72.2 185.2  84.3 143.9  74.4

0.017

FUC MAN

MAN (Table I), which suggest similar dilution of the exoglycosidases activity in the nasal polyps tissue. Of exoglycosidases tested in nasal polyps only concentration activity of MAN (taking part in degradation of N-linked oligosaccharides) did not significantly decrease, which suggest weaker involvement of N-linked glycoproteins in nasal polyps pathology. Full inflammatory response includes production of the inflammation mediators such as prostaglandins [34] and cytokines [35] responsible for local inflammatory reaction. Decrease in concentration of exoglycosidases and presence of some inflammatory cells in nasal polyps, i.e. acidophilic and neutrophilic granulocytes suggest deregulation of the mechanisms of inflammation. In full symptomatic inflammatory processes, e.g. tonsillitis [38] was reported increase in tissue remodeling expressed by increase in lysosomal catabolism of glycoconjugates, reflected by increase in concentration of activity the lysosomal exoglycosidases. Decrease in concentration of exoglycosidases activity participating in the catabolism of oligosaccharide chains of glycoconjugates in nasal polyps tissue (Figs. 2 and 3) is an argument against recognition nasal polyps as a fully inflammatory disease. Our results confirm inflammatory-bioelectrical theory of nasal polyp pathogenesis [17, 27, 28]. Our results corroborate the decrease in secretion of cytokines, expression of adhesion molecules, amount of eosinophiles and mastocytes observed in polyps tissue [39–43]. Glucocorticosteroid application in prevention of nasal polyps recurrence suggests involvement of chronic inflammatory immunological mechanism deregulation in pathogenesis of the nasal polys [44, 45]. Our results agree with results of Metzler et al. [46], that inflammation precedes nasal polyps, by accumulation of fluids and albumins in subepithelial layer. RostkowskaNadolska et al. [45, 47] reported that cytokeratines 4,13, and 19 expressions suggest involvement in nasal polyps rather stroma than epithelium. Our results as well as lack of cells edema in nasal polyps tissue [18], and excessive hydratation of nasal polyps extracellular matrix are in agreement with

0.007 0.072

Bernstein and Yankaskas suggestion [48, 49], that one of the factors responsible for nasal polyps creation are disturbances in transport of water and electrolytes in nasal mucosa. Bernstein and Yankaskas proposed the abnormal Cystic Fibrosis Transmembrane Regulator (CFTR) protein, regulating sodium channels (ENaC), for increase in amount of open sodium channels on the surface of nasal mucosa endothelial cells and stromal swelling [48]. For absorption of sodium cations in stroma also Major Basic Proteins may be responsible, secreted by eosinophils, which decrease secretion of mucus and increase absorption of sodium [17, 22]. Higher ability to absorb sodium and chloride ions by nasal polyps mucosa supports the opinion of significant role of deregulation water and mineral metabolism in pathogenesis of nasal polyps [27]. In extracellular matrix main substances responsible for water absorption are hyaluronate and proteoglycans (e.g. chondroitin sulfate), because of their negative charged polyanion and hydrophilic –OH groups [50]. It is worthy of note that only small part of water molecules are directly bound to the proteoglycans by hydrogen bounds, but majority of water molecules is mechanically trapped inside of heteropolysaccharide structure of hyaluronate [51, 52]. Therefore it would be a valuable information on composition and distribution of glycosaminoglycans and proteoglycans in nasal polyps tissue in comparison to normal nasal mucosa. In conclusion, our research supports bioelectrical theory of nasal polyps pathogenesis and directs attention at research on glycoconjugates (particularly glycosaminoglycans) of the nasal mucosa extracellular matrix, the structural elements responsible for absorption the excessive amounts of water.

Authors' contributions/Wkład autorów SC – study design, data collection and interpretation, acceptance of final manuscript version, literature search. AM – study design, data collection and interpretation, acceptance

Table II – Proportions of lysosomal exoglycosidases specific activities in nasal polyps and control tissues Exoglycosidase

Tissue

N

Specific activity (pKat/mg protein)

p

GAL

control polyp control polyp control polyp

20 40 20 40 20 40

83.3  24.9 73.6  45.6 65.1  27.8 51.5  31.0 58.9  22.2 54.3  36.2

0.292

FUC MAN

0.093 0.544

196

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of final manuscript version. MK, AN-J – data collection and interpretation, acceptance of final manuscript version. PK – statistical analysis, data interpretation, acceptance of final manuscript version. BZ-S – data collection, acceptance of final manuscript version. AK, NW – data interpretation, acceptance of final manuscript version. ŁM – statistical analysis, data interpretation, literature search. KZ – study design, data interpretation, acceptance of final manuscript version, funds collection. SDS – study design, data interpretation, acceptance of final manuscript version.

Conflict of interest/Konflikt interesu None declared.

Financial support/Finansowanie The study was financed from the grant of Medical University of Bialystok No. R-I-002/46/2007.

Ethics/Etyka The work described in this article have been carried out in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans; EU Directive 2010/63/EU for animal experiments; Uniform Requirements for manuscripts submitted to Biomedical journals.

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