Nasal fluid secretory immunoglobulin A levels in children with allergic rhinitis

Accepted Manuscript Title: Nasal fluid secretory immunoglobulin A levels in children with allergic rhinitis Author: Fatih Dilek Emin Ozkaya Bilge Gultepe Mebrure Yazici Meryem Iraz PII: DOI: Reference:

S0165-5876(16)00031-8 http://dx.doi.org/doi:10.1016/j.ijporl.2016.01.018 PEDOT 7936

To appear in:

International Journal of Pediatric Otorhinolaryngology

Received date: Revised date: Accepted date:

10-10-2015 18-1-2016 19-1-2016

Please cite this article as: F. Dilek, E. Ozkaya, B. Gultepe, M. Yazici, M. Iraz, Nasal fluid secretory immunoglobulin A levels in children with allergic rhinitis, International Journal of Pediatric Otorhinolaryngology (2016), http://dx.doi.org/10.1016/j.ijporl.2016.01.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nasal fluid secretory immunoglobulin A levels in children with allergic

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rhinitis

Fatih Dileka*, Emin Ozkayaa, Bilge Gultepeb, Mebrure Yazicia , Meryem Irazb

Department of Pediatric Allergy and Immunology, Bezmialem Vakif University Medical

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a

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Faculty. b

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Running title: Secretory Ig A levels in allergic rhinitis.

Department of Clinical Microbiology, Bezmialem Vakif University Medical Faculty,

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Istanbul, Turkey.

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Conflict of interest: None.

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Institution: Bezmialem Vakif University.

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This work was supported by a Bezmialem Vakif University grant. The study was approved by the Bezmialem Vakif University Ethical Committee (71306642/050-01-04/169).

Correspondance to: Fatih Dilek MD. Department of Pediatric Allergy and Immunology, Bezmialem Vakif University Medical Faculty, Adnan Menderes Bulvari Vatan Caddesi 34093 Fatih/Istanbul . Tel: +902125232288; fax: +902124531870; E-mail: [email protected].

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ABSTRACT Objectives: There is growing knowledge about the immunoregulatory and possibly preventative roles of immunoglobulin A (IgA) in allergic diseases. This study aimed to

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investigate secretory immunoglobulin A (SIgA) levels in the nasal fluid of children who were either being treated for their allergic rhinitis (AR) with intranasal mometasone furoate or were

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not receiving treatment.

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Methods: The study population contained 55 children with persistent AR. Group I included 27 newly diagnosed AR patients not taking any medication and group II included 28 patients

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treated with intranasal steroids for at least 6 months. 27 healthy control subjects were also enrolled in the study. Total symptom scores (TSS) were calculated for each patient. Nasal

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secretions were obtained using a new modified polyurethane sponge absorption method, and

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samples were analysed by ELISA.

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Results: The median value for nasal fluid SIgA level in each group was 127.2 µg/ml (interquartile range; 67.3-149.6) in group I, 133.9 µg/ml (102.1-177.8) in group II and 299.8

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µg/ml (144.5-414.0) in the control group. Groups I and II both had statistically significant reductions in nasal fluid SIgA levels compared to the control group (p<0.001). However, there was no statistically significant difference between groups I and II (p=0.35). A statistically significant and negative correlation also existed between TSS and nasal fluid SIgA levels in both groups I and II (p=0.006, rho= -0.512 and p=0.01, rho=-0.481, respectively).

Conclusions: SIgA levels in the nasal fluid are significantly reduced in children with AR independent of treatment and are negatively correlated with the TSS. Key words: Allergic rhinitis; treatment; children; secretory Ig A; nasal fluid; polyurethane.

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1.Introduction Allergic rhinitis (AR) is characterised by sneezing, rhinorrhea, nasal congestion, itching and eye symptoms [1]. According to a large multicentre study, the overall prevalence of AR in

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children aged 6–7 and 13–14 was 8.5% and 14.6%, respectively [2]. Mucosal surfaces are the main gateway for antigens. The local production of secretory immunoglobulin A (SIgA)

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composes the primary component of mucosal immunity [3]. SIgA is the major immunoglobulin present on mucosal surfaces and it has pronounced antimicrobial effects.

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epithelial adherence and invasion by pathogens [4].

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SIgA performs bacterial agglutination, endotoxin and virus neutralisation, and it can impede

Alongside, there is an increased body of literature about the immunoregulatory roles of SIgA.

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Allergic disorders appear to be common in patients with Ig A deficiency [5]. The bronchoalveolar lavage fluid SIgA contents of asthmatic patients are decreased compared to

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healthy controls, and there was a correlation between spirometric values and asthma

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symptoms [6,7]. High salivary SIgA levels were associated with a lesser development of allergic symptoms in sensitised children [8]. In animal models, a lack of secretory antibodies

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may cause immune dysfunction and intolerance against foods [9]. The administration of cholera toxin B, which is a mucosal adjuvant, to the lungs induces the production of IgA, thereby suppressing the clinical and laboratory features of asthma [10]. Intranasal corticosteroids (INSs) are a mainstay of AR treatment, and their effects on nasal symptoms are well described [11]. But, some studies showed that even short-term systemic corticosteroid treatment can cause prolonged reductions in serum immunoglobulin G and A levels [12,13]. However, there is not enough data about the effects of INSs on nasal SIgA levels in children. Our literature search reveals there are insufficient and conflicting results about nasal fluid SIgA levels in AR patients and no studies about whether INSs affect nasal fluid total SIgA concentrations in children. Therefore, we aimed to measure SIgA levels

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in the nasal fluids of pediatric AR patients, both newly diagnosed and those taking regular INS treatment, to investigate the possible role of SIgA in the pathogenesis of AR, as well as the effects of steroid treatment on nasal fluid SIgA levels.

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2. Matherials and methods

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2.1. Patients

This study was designed with a case-control study format and performed in the Bezmialem

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Vakif University Pediatric Allergy and Immunology Department between July 2014 and

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January 2015. In total, 216 consecutive patients with persistent AR were evaluated for their eligibility to enrol in the study. Inclusion criteria were (1) age between 6 and 18 years; (2) AR

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diagnosis established according to criteria defined in the Allergic Rhinitis and its Impact on Asthma (ARIA) guidelines [14]. Exclusion criteria were (1) acute or chronic infectious or

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inflammatory diseases (including asthma and atopic dermatitis); (2) history of maternal or

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paternal smoking; (3) use of any medications except for mometasone furoate (Nasonex®, Merck, NJ, USA) including other INSs, oral or inhaled corticosteroids, antihistamines,

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montelukast, poly-vitamins or minerals; (4) treatment period of less than 6 months, or dosage different than 100 mcg/day if patients taking mometasone; (5) non-compliance with mometasone therapy.

The reason we chose to conduct this study in patients more than 6 years of age was to be able to perform lung function studies and to facilitate the tolerance of the nasal sample collection procedure. To attempt to eliminate possible confounding factors related to the pharmacological properties of drugs and dosage regimens, we created a homogeneous study group that took the same dosage of the same drug. We chose mometasone furoate for the INS because it is the most commonly prescribed INS in our country. Some reports have suggested that asthma, smoking and vitamin supplementation may affect SIgA levels and its expression

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in some biological fluids; therefore, all these factors are considered exclusion criteria [7,1517]. All AR patients were evaluated using a standard questionnaire in terms of the exclusion criteria and drug usage mentioned above. Patients who had asthma that was diagnosed by a

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doctor or signs or symptoms consistent with asthma or abnormal lung function studies were also excluded from study. The clinical diagnosis of asthma was determined using the criteria

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defined in the Global Initiative for Asthma guidelines [18]. The most common reasons for exclusion from the study were presence of asthma (59 patients) and, use of other medications

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(48 patients), maternal or paternal smoking (11 patients), presence of atopic dermatitis (9 patients), non-compliance with mometasone treatment (13 patiens), different dosing regimens

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or shorter treatment duration (5 patients). Seventy-one patients in total met the criteria and

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enrolled in the study. Seven patients whose parents refused permission for their participation in the study, 5 patients who could not tolerate the nasal secretion sampling procedure, and 4

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patients whose nasal secretions could not to be obtained even with proper application of the

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procedure were excluded from the study despite initially meeting the exclusion criteria. After this exclusion, the total number of the study participant was determined as 55.

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Group I consisted of 27 patients newly diagnosed with persistent AR who had never before received therapy, and group II consisted of 28 patients with persistent AR who had been regularly taking intranasal mometasone furoate at a dose of one puff per nostril once a day (total: 100 µgr/day) as monotherapy for at least 6 months. These patients had been attending the same outpatient clinic regularly every 2 months. Compliance with the dosing regimen was assessed every 2 months through observation of the nasal spray devices and dose calculations as well as through each patient’s mother signing a diary card indicating that the drug had been administered. If the drug was not taken for more than 10% of the scheduled doses, the patient was considered non-compliant with therapy and excluded from the study.

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The control group consisted of 27 healthy children who periodically attended pediatric clinics at the same hospital for regular developmental check-ups. Children were included in the control group if they had no history of any allergic disease or paternal or maternal smoking

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and were not taking any other medications, including vitamins, minerals and analgesics. Children enrolled in the study also had no signs or symptoms of acute or chronic infectious or

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inflammatory disorders. The study was performed in accordance with the Declaration of Helsinki Good Clinical Practice guidelines and was approved by the Bezmialem Vakif

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University Ethical Committee (71306642/050-01-04/169). Informed consent was obtained

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from the parents of all children. 2.2. Collection of nasal secretions

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Nasal secretions were obtained using a polyurethane sponge nasal secretion collector (NSC) according to the method described by Lü et al. but with minor modifications [19]. A sixty

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pores per inch reticulated polyurethane sponge was cut into rectangular prism shapes (base of

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5×10 mm and height 20 mm) using a homemade cutting device, and the pieces were sterilised

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by autoclaving for 20 min at 121 °C prior to use. A single-use metal rod with a tip clamp was used to hold the sponge during sample collection (Fig. 1.). The sponge was inserted and placed on the floor of the nasal cavity between the septum and the inferior turbinate for at least 5 min. The sponge containing nasal secretions was pulled out from the nostril and inserted into an inner tube that was in the outer centrifuge tube (Fig. 1). We pierced the bottom of a 2 ml Eppendorf tube (Eppendorf AG, Hamburg, Germany) using a 21-gauge needle and created 15 standard holes to allow nasal secretions to pass into the outer tube during centrifugation. The outer tube was a standard 10 ml vacuum blood collection tube. Next, this device was centrifuged at 3,000 g for 10 min to recover the nasal fluid. We obtained a median volume of 300 microlitres (µl) of nasal fluid from patients and control

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subjects (interquartile range [IQR]; 200-450 µl) using this method. The extracted fluid was stored at -80 °C until assayed. 2.3. Measurement of SIgA levels

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SIgA levels in nasal fluid samples were measured using a secretory Ig A ELISA kit

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(Salimetrics, Suffolk, UK). Samples were thawed at room temperature and then centrifuged at 3,000 rpm for 10 min. Samples were diluted to obtain the appropriate concentrations, and an

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ELISA was performed according to the manufacturer’s instructions. Supplied standards were used to generate the standard curves, and the samples and standards were added to the wells.

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Unbound protein was removed by washing, and conjugate was added. After a colour reaction with a substrate, the optical density was recorded using an automated ELISA reader at a

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wavelength of 450 nm. The absorbance at 450 nm was converted to µg/ml, and the minimal

2.4. Nasal symptoms

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detection limits were 2.5 g/ml.

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The same pediatric allergist (FD) evaluated AR symptoms in the study population using the

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total symptom score (TSS) scale (maximum score, 12). Total symptoms scores TSSs were calculated in interviews with patients and families using the sum of the scores for nasal obstruction, rhinorrhea, sneezing, and nasal itching. Each nasal symptom was scored from 0 to 3 (0= no symptoms; 1= mild; 2= moderate, 3= severe). Symptoms were scores as ‘mild’ if they were clearly present but easily tolerated. A ‘moderate’ score was given if symptoms caused discomfort but did not interfere with daily activity or interfere with patients’ sleep. Symptoms that interfered with daily activities and sleep were scored ‘severe’ [20,21]. 2.5. Skin prick test Skin prick tests were performed using Stallerpoint® lancets and allergen solutions manufactured by Stallergenes (Stallergenes, Paris, France). In total, 15 different aeroallergens,

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including house dust mites, grass, tree pollens, fungi and animal dander, were tested according to the Global Allergy and Asthma European Network's suggestions [22]. Skin prick tests were considered positive if the presence of a wheal of a maximum diameter of 3 mm

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remained once the negative control value had been subtracted.

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2.6. Lung function studies

Spirometry was performed before and after administration of 200 µgr salbutamol, admistered

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with Able spacer® (Clemente Clarke, Harlow, England) according to European Respiratory Society guidelines [23]. Forced expiratory volume in one second (FEV1) rates were measured

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with dynamic spirometry (Vitalograph®; G.W. Berg-Co., UK). The best of 3 successful manoeuvres was recorded. Reversibility was defined as the presence of a >12% change in

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FEV1 after salbutamol administration [18].

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2.7. Statistics

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A statistical analysis was performed using IBM SPSS 19 (IBM, Armonk, NY, USA). A

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Shapiro–Wilk test was used to test distributions for normality. Parametric data were expressed as the mean ± standard deviation (SD), and non-parametric data were expressed as the median, IQR. The Kruskall Wallis test was used to compare more than two independent parameters and a Mann–Whitney U test was used to calculate the difference between two parameters in groups. Correlation between two variables assessed by using the Spearman rank correlation coefficient. Categorical data were evaluated using the chi square test and p < 0.05 was accepted as statistically significant. 3. Results 3.1. Patients

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Some demographic and clinical data for the study and control groups are shown in Table 1. The median ages were 10.0 years (IQR; 7.5-14.3 years) in group I, 9.4 years (7.1-11.5) in group II, and 10.3 years (8.5-13.3) in the control group. The male: female ratio of the study

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and control groups were 18:9, 17:11 and 17:10, respectively. No significant differences between the groups existed with respect to age and gender (p>0.05). The median duration of

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mometasone treatment was 8 months (7-10) in group II.

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3.2. Skin prick test and lung function studies

Monosensitization to mites was the most frequently detected aeroallergen sensitivity in both

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AR groups (89% in group I and 86% in group II; Table 1). In total, six patients had multiple aeroallergen sensitization (to mites and grass [n=3], to mites and trees [n=2], and to mites and

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cats [n=1]). Nasal fluid samples of pollen-sensitized patients were obtained outside the pollen season. Mean FEV1 values of the groups were 91.5% ± 10.2 and 89.1% ± 5.6 of predicted

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values, respectively; reversibility was not detected in any of the patients. No statistically

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(p>0.05; Table 1).

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significant differences existed between groups I and II in terms of these clinical features

3.3. Nasal symptoms

The median value of TSS in groups I and II was 7 (6-8) and 3 (2-4), respectively. A statistically significant difference existed according to TSSs between the 2 groups (p<0.001; Table 1).

3.4. Nasal fluid SIgA levels The median values of nasal fluid SIgA levels were 127.2 µg/ml (67.3-149.6) in group I, 133.9 µg/ml (102.1-177.8) in group II and 299.8 µg/ml (144.5-414.0) in the control group. Both groups I and II have statistically significant reduced SIgA levels compared to the control

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group (p<0.001; Fig. 2). However, there was not a statistically significant difference between groups I and II (p=0.35; Fig. 2). 3.5. Correlations

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A statistically significant negative correlation existed between TSS and nasal fluid SIgA levels in both groups I and II (p=0.006, rho=-0.512 and p=0.01, rho=-0.481, respectively; Fig.

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3).

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4. Discussion

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Our study results showed that children with AR have decreased total nasal fluid SIgA levels independent of treatment. The polyurethane method is easy to use, tolerable, non-invasive and

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has a higher detectability and reproducibility than nasal lavages for analysing immunological markers in nasal secretions; therefore, it is the preferred method for studies like ours [19]. To

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polyurethane NSC method.

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our knowledge this is the first study conducted in a pediatric AR population using the

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We also detected a statistically significant negative correlation between nasal fluid SIgA levels and TSSs in both AR groups. The differences in terms of TSSs was also statistically significant between groups I and II. These findings are plausible because group I consisted of newly diagnosed, untreated and highly symptomatic children, while group II consisted of patients who had taken mometasone furoate 100 µgr/day as monotherapy for a long time. The individuals in group II had benefited from treatment for a considerable period of time and were minimally symptomatic, and therefore did not require additional medications or higher doses. Our literature search did not find any studies that examined the relationship between severity of AR symptoms and nasal fluid SIgA levels. As was described earlier in the study of Balzar et al., SIgA levels in the bronchoalveolar lavage fluid of individuals with asthma correlated positively with lung function studies and negatively with asthma symptoms [7].

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Tsitoura et al. reported that B cells critically influence the immune response to inhaled allergens [24]. Reduced or altered repertoire within SIgA could result in inappropriate or allergic reactions against aeroallergens [7]. Although further studies focusing on AR are

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needed, according to the concept of “one airway, one disease” it is very likely that the information we obtained from asthma studies is valid for AR. Based on our study results, not

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only is reduction in local SIgA production a component of dysregulated mucosal immunity, it

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may be one of the determining indicators for AR symptom severity.

In a previous study, Hsin et al. stated adult AR patients have similar nasal fluid SIgA levels

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compared to healthy subjects [25]. In this study, the lavage method was used to collect nasal secretions. This is not the preferred method for nasal sample collection at the present time;

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therefore, methodological problems may explain the differences between our results. Hupin et al. identified that the epithelial polymeric Ig receptor expression is decreased and there are

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subepithelial accumulations of IgA in sinonasal biopsy specimens of adult patients with AR.

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However, the levels of total IgA and Ig A subclasses in nasal secretions were not significantly different from the control group [26]. However, Only 13 AR patients were participated in this

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their study, and the small sample size may make it difficult to demonstrate differences between groups statistically. Additionally, there were smokers in both the control and AR groups. This factor may have affected the results; while no data exist in the literature for nasal fluid SIgA levels, it is known that smoking may decrease IgA levels in saliva [15]. Cortesina et al. demonstrate the titration of SIgA in the nasal secretions of AR patients, as determined by the immunoisoelectric-focusing method, significantly decreased compared to the control group [27]. To investigate inflammation at the site of the disease process, the most direct way is to measure cytokine levels from the mucosa. However, a mucosal biopsy is an invasive procedure associated with patient discomfort, tissue injury and possible scar formation. In

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addition, there is good evidence that nasal secretion cytokine levels are correlated with the clinical course and sinus biopsy results [28,29]. Nasal secretion sampling techniques can be categorized in (1) collection of spontaneous secretions, (2) dilution techniques and (3)

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absorption techniques [30]. The collection of spontaneous secretions (e.g. nose blowing, suction) is feasible only in patients with nasal hypersecretion, but not in healthy subjects.

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Nasal lavage is the dilution technique most frequently used; however, during application, the swallowing or absorption of unknown fractions of lavage fluid is possible. The substantial and

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typically unpredictable dilution of nasal secretions is related to these techniques [30]. In a study by Lü et al., the concentrations of cytokines, eosinophil cationic protein and tryptase in

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nasal secretions obtained using polyurethane NSCs were at least 8-fold higher than those

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tested in nasal lavages were. Furthermore, the levels of immunoglobulins and allergenspecific antibodies showed a 6–290-fold increase when the NSC was used [19].

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The highest prevalence of selective Ig A deficiency reported in a healthy population is 1/328,

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and there are well-documented reports of patients with normal nasal SIg A levels despite serum Ig A deficiency [31-33]. Therefore, we did not evaluate serum Ig A levels, instead

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focusing only on nasal secretory Ig A levels, and none of the patients or control subjects had extremely low or undetectable SIgA levels. Asthmatic patients treated with an inhaled corticosteroid + long-acting β- agonist combination or an inhaled steroid alone had no significant variation in their salivary SIgA levels [34,35]. Two clinical trials conducted on AR patients showed intranasal budesonide treatment does not reduce nasal fluid allergen-specific Ig A levels [36,37]. All these findings show that topical steroids do not alter the Ig A content of biological fluids and these findings are compatible with our results.

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Obviously, perfoming this study in a randomized, controlled manner would have been preferable to clearly demonstrate the effects of INSs on SIgA levels in nasal fluid; this is one limitation of our study. However, we cannot afford stringent requirements and ethical issues

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of prospective drug studies in our country. For this reason, a prospective study could not be undertaken. In lieu of this, we planned the study as a case-control study. If we had obtained

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pre- and post-nasal fluid SIgA levels from the mometasone treated group (group II), or could re-study results of these parameter at group I, our results would be more rigorously tested and

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impressive in terms of the effect of INSs on SIgA levels. However, we studied nasal fluid SIgA levels in a newly diagnosed, treatment-naïve group in order to provide a comparison to

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the group taking mometasone for at least 6 months (group II). Results taken from group I

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provide sufficient idea about the pretreatment status of group II. On the other hand, our study results are inadequate to take the next step and suggest that INSs do not alter nasal fluid SIgA

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levels. We also want to highlight that this is a pivotal study and larger prospective studies are

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5. Conclusions

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needed on this issue.

In our study, SIgA levels in nasal fluid were significantly reduced in children with AR compared to healthy subjects and were negatively correlated with the TSS. In addition, SIgA levels are not different between the groups taking and not taking INS treatment. Our findings draw attention to the role of IgA in the pathogenesis of AR. Conflict of interest statement None. Acknowledgement This work was supported by a Bezmialem Vakif University grant.

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30. H. Riechelmann, T. Deutschle, E. Friemel, H.J. Gross, M. Bachem, Biological markers in nasal secretions, Eur. Respir. J. 21 (2003) 600-605.

31. J.A. Clark, P.A. Callicoat, N.A. Brenner, C.A. Bradley, D.M. Smith, Selective IgA deficiency in blood donors, Am. J. Clin. Pathol. 80 (1983) 210-213.

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32. P.J. Stanley, P.J. Cole, The concentrations of IgA and free secretory piece in the nasal secretions of patients with recurrent respiratory infections, Clin. Exp. Immunol. 59 (1985) 197-202.

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review of the literature, Medicine 50 (1971) 223-236.

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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. 106 (2000) 521-529.

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No decrease of birch pollen

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Fig. 1. Picture of nasal sample collector.

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Fig. 2. Nasal fluid secretory immunoglobulin A (SIgA) levels in the study and control groups. The box plots span data values between the first and third quartiles. The dark horizontal line

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the 75th and 25th persentiles ±1.5 times the interquartile range.

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within the box represents the median. The upper and lower fences represent the value equal to

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Fig. 3. Negative correlation between nasal fluid secretory immunoglobulin A (SIgA) levels

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and total symptom score (TSS) in group I (A), and group II (B).

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Table 1 Some demographic and clinical features of the study and control groups. Group II

Control group

(n = 27)

(n = 28)

(n = 27)

(7.5- 9.4 (7.1-11.5) 10.3 (8.5-13.3)

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10.0

Age (years, median, [IQR])

18/9

TNSS TSS (median, [IQR])

7 (6-8)

17/10

3 (2-4)

Mites

24 (%89)

24 (%86)

2 (%8)

Fungi

1 (%3)

-

3 (%11)

3 (%11)

-

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-

INS treatment duration (months, -

> 0.05

> 0.05 <0,001

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Aeroallergen sensitivity (n [%] )

17/11

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Sex (male/female)

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14.3)

P value

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Group I

8.0 (7.0-10.0) -

median, [IQR])

IQR, Interquartile range; SD, Standart deviation; TNSS, Total nasal symptom score TSS, Total symptom score; INS, Intranasal steroid.

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Figure1

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Figure1black-white

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Figure2

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Figure3

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