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ORIGINAL ARTICLE
Iran J Allergy Asthma Immunol
December 2010; 9(4): 225-230.
Effect of Treatment with Intranasal Corticosteroid and Oral Antihistamine on
Cytokine Profiles of Peripheral Blood Mononuclear Cells of Patients with
Allergic Rhinitis Sensitive to Chenopodium album
Shokrollah Farrokhi1,2, Tahereh Mousavi1,3,4, Saba Arshi5, Naser Javahertarash5, Abdolreza Varasteh6,
Reza Falak6, Nima Rezaei7, Alireza Salekmoghadam1,4
1 Department of Immunology, Tehran University of Medical Sciences, Tehran, Iran 2 Department of Immunology, Bushehr University of Medical Sciences, Bushehr, Iran 3 Microbial Resistance Research Center, Tehran University of Medical Sciences, Tehran, Iran 4 Immunology Research Center, Tehran University of Medical Sciences, Tehran, Iran 5 Department of Allergy & Clinical Immunology, Rasoul Akram Hospital, Tehran University of Medical Sciences, Tehran, Iran 6 Immunobiochemistry Lab, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad, Iran 7 Molecular Immunology Research Center; and Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran Received: 20 April 2009 ; Received in revised form: 12 June 2009 ; Accepted: 1 November 2009
ABSTRACT
Patients with al ergic rhinitis (AR) show increased production of the Th2-related cytokines. Almost always, intranasal corticosteroid (INC) and antihistamine are used as routine therapy of AR. This study was performed to determine the in vitro secretion of cytokines profiles of PBMCs in patients with AR sensitive to Chenopodium album (Ch.a) pollens before and after treatment with INC (Fluticasone propionate) and oral antihistamine (Loratadine). PBMCs of 20 patients with AR, were tested in vitro for cytokine production. These cells were stimulated with natural or recombinant Ch.a. The levels of IL-4, IL-13 and IFN-, were measured in supernatants of cultured cel 96h after stimulation using ELISA. The PBMCs of 20 normal individuals were also similarly treared for comparison of results. The production of IL-4 by the patients’ cells stimulated with either Ch.a or rCh.a was significantly higher than normal levels before therapy (p=0.04 and p=0.02, respectively). After therapy, a significant decrease in production of IL-4 and a significant increase in production of IL-10 were found in PBMCs stimulated with natural Ch.a, in comparison to the results before stimulation (p=0.03 for IL-4; p=0.04 for IL-10). Similarly, these results were seen in the production of IL-4 and IL-10 stimulated with rCh.a al ergen after therapy in comparison to the results before stimulation (p=0.01 for IL-4; p=0.03 for IL-10). This study suggests INC (Fluticasone propionate) and oral antihistamine (Loratadine) have the capacity to inhibit the production of IL-4 and shift Th2/Th1 responses, probably due to increase the level of immunoregulatory IL-10. Therefore, it could be concluded that therapy with INC and antihistamine has pharmacologic and immunologic therapeutic effects on AR patients.
Key words: Allergic Rhinitis; Antihistamine; Cytokines; ELISA; Intranasal Corticosteroid; PBMCs
Corresponding Author: Tahereh Mousavi, PhD;
Medical Sciences, Tehran, Iran. Tel/Fax: (+98 21) 8805 8652, Department of Immunology, Medical College, Iran University of Copyright 2010, IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY. All rights reserved. INTRODUCTION
MATERIALS AND METHODS
Allergic rhinitis (AR) is a chronic IgE-mediated Subjects
inflammatory disease, which is triggered by allergens, Twenty patients with AR were enrolled in this including seasonal allergens such as weeds, pollen. The study. Demographic data of all subjects are shown in prevalence of AR is growing up worldwide and it is table 1. The inclusion criteria were subjects with age estimated that up to 20% of general population are ≥18 years, presence o f rhinitis symptoms, affected by AR.1 Chenopodium album (Ch.a) is one of the most allergenic plants with high abundance growth allergen (Stallergen, France).13 We excluded subjects in most parts of Iran.2 At present, Ch.a natural extracts with more than 50 years who did not participate in are used for diagnosis and immunotherapy of AR second time sampling, and had high titer of total IgE patients. The natural homemade extract of Ch.a has due to nonallergic causes and 20 normal individuals been prepared and examined in skin prick test in a with no history of AR and a negative skin prick test (SPT) to the Ch.a allergen. The patients were classified Also, Mousavi et al induced asthma in animal according to the ARIA classification.7 All participants model using homemade natural extract of Ch.a which resulted in increasing the levels of IL-4 and IL-5 study. INC (Fluticasone propionate, 2 puff, morning, as well as specific Ch.a IgE in mice.4 It has been gsk, UK) and antihistamine (Loratadine, 10 mg, orally, suggested that increased activity of T-helper (Th)2 daily, Razi co, Iran) had been prescribed for all patients cells, which produce IL-4, IL-13 and IL-5 cytokines as routine therapy. Blood samples were collected contribute to allergic inflammation.5,6 Intranasal before starting therapy at first visits and 6 weeks later corticosteroid (INC) and antihistamine are introduced during pollination season of Ch.a (late summer and as the first line of therapy for AR by the “Allergic fall). The study was approved by the local ethics Rhinitis and its Impact on Asthma (ARIA)” global committee. Written informed consents were also Several studies showed that INC reduced the level of cytokines secreted by Th2 cells and inhibited levels Study Design
of other cytokines such as IL-1β, IL-8, GM-CSF and Five milliliters (ml) of venipuncture blood from all TNF-α in AR.8-12 These reports indicate that subjects were collected into falcon tubes containing maintenance treatment with INC can have anti- EDTA (EthyleneDiamineTetraacetic Acid). PBMCs were immediately isolated using Ficoll-Paque The main goal of this project was, without (Fresenius Kobi Noge As for Axis-Shield Poc As, Oslo, intervention in the usual treatment of the patients with Norway) and gradient centrifugation. Isolated PBMCs AR, to study the effects of intranasal corticosteroid and were cultured in a 24-well plate (JET Biofil®, Canada) oral antihistamine on cytokine production of PBMCs of in RPMI 1640 (Biosera, UK) containing 10% fetal calf serum (FCS) (Biosera, UK), 50 µg/ml penicillin, 50 Moreover, we studied cytokine profiles of Th1, Th2 µg/ml streptomycin (Biosera, UK), 0.3 mg/mL L- and immunoregulatory cytokines after therapy of AR glutamine (Biosera, UK), at a density of 1×106 cells per well and incubated (at 37°C and in 5% CO2) in the For this, we explored the effects of combination use presence of 50 µg/ml natural Ch.a or 10 µg/ml rCh.a, of INC (Fluticasone propionate) and antihistamine separately. Supernatants were collected before and after (Loratadine) on the Th1 cytokine [with assay of therapy of patients and aforementioned cytokines were Interferon (IFN)-γ], Th2 cytokines (with assay of IL-4 and IL-13) and immunoregulatory cytokine (with assay Natural Ch.a allergen was extracted and purified of IL-10) profiles secreted by peripheral blood from whole Ch.a pollen as described before.3 rCh.a mononuclear cells (PBMCs) isolated from patients with allergen was prepared in Immunobiochemistry Lab, AR and from normal individuals exposed to natural or Immunology Research Center, Avicenna Research recombinant Ch.a (rCh.a) allergens in vitro. Institute, Mashhad University of Medical Sciences, 226/ IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Effect of Intranasal Corticosteroid and Oral Antihistamine Treatment on Allergic Rhinitis Table 1. Subjects’ Characteristics
Features
Allergic rhinitis
Normal control
patients
Skin Prick Test (SPT), Allergic Rhinitis and Its Impact on Asthma (ARIA) Levels of IFN-γ, IL-4, IL-10, and IL-13 were baselines obtained from normal individuals. There was measured in supernatants 96 h after culture, using also no significant difference on IFN-γ production in ELISA sets from U-CyTech biosciences (Utrecht, The Netherlands) with sensitivity of of 2 pg/ml. Total IgE were also measured with ELISA kit in sera of all AR Effect of INC and Antihistamine on IL-4 and IL-13
patients with sensitivity 1 IU/l (PADTAN ELM, Iran). Production
PBMCs of AR patients with moderate- severe Statistics
persistent AR (10 cases) secreted significantly higher Statistical analysis was performed using SPSS 16 IL-4 in comparison with PBMCs of other AR patients software package (San Diego, CA). Comparisons of the results before and after treatments were also done using To investigate the inhibitory effects of INC and Wilcoxon Signed Ranks Test. P <0.05 were considered antihistamineon, the cytokine secreted in response to Ch.a allergens by PBMCs, including IL-4 and IL-13 in supernatants were measured. Stimulated PBMCs from patients with AR secreted significantly higher IL-4 in supernatants after exposure to natural Ch.a or rCh.a, As shown in table 1, there was no significant compared to normal group (P=0.04 and P=0.02, difference between these two groups with respect to respectively), whereas after therapy of AR patients, the gender distribution and age. Compared with the level of IL-4 in supernatant of their PBMC decreased patients, normal subjects were negative for allergen significantly. (P=0.03 and P=0.01, respectively). skin test and did not show any clinical symptoms of AR Effect of INC and Antihistamine on IL-10
(Immunoregulatory Cytokine) Production
Effect of INC and Antihistamine on IFN-γ
In order to demonstrate whether IL-10 could be an Production
immunoregulatory cytokine to inhibit Th2 responses, The levels of secreted of IFN-γ by natural Ch.a or we measured IL-10 and found that secreted IL-10 in rCh.a-stimulated PBMCs from patients before and after supernatants after therapy of patients was significantly treatments and normal individuals are shown in table 2. increased, after exposure to natural Ch.a or rCh.a There was not any significant difference on IFN-γ allergens (P=0.04 and P=0.03 for both allergens, production before therapy of patients compared to the IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY /227 Table 2. Cytokine profiles of natural or recombinant Ch.a (rCh.a) stimulated PBMCs from patients with AR before and
after treatments in comparison with stimulated PBMCs from normal control.
Cytokine
PBMCs exposed to natural Ch.a
PBMCs exposed to rCh.a
AR patients
AR patients
P value
P value
controls
controls
treatment
treatment
treatment
treatment
Chenopodium album (Ch.a), Peripheral Blood Mononuclear Cells (PBMCs), Allergic Rhinitis (AR) Values are mean ± standard deviation (SD) *P< 0.05 compared with normal control group DISCUSSION
cells. Similarly, our data indicated that the production of IL-4 by PBMCs of AR patients was significantly Although INC and antihistamine are commonly decreased. But IL-10 in supernatants was increased used in the treatment of AR, at present the after therapy with INC and antihistamines. According immunomodulatory effects of these drugs on immune to Wolk et al.25 who indicate that IL-10 is required for antigen presentation to regulate the nature and extent of Allergic reaction inducing AR symptoms can occur the T helper response, we concluded that increase in early and late phases. The late phase reaction occurs production of IL-10 induced by INC and antihistamine 6 to 24 hrs after exposure to an allergen, which is may could regulate the expression of IL-4 by PBMCs. associated with cellular infiltration of the nasal mucosa Sim et al.26 also showed that treatment of patients with with activated CD4+ Th2 cells and eosinophils.14 topical decongestants, as vasoconstrictor, selectively Recent studies showed that cytokines IL-4, IL-5, IL-9 induced decrease of IL-5. This study confirms and IL-13 play a crucial role in this inflammatory synergistically effect of combination therapy of INC reaction,15,16 while IFN-γ and IL-10 have been and antihistamine in patients with AR which enhanced concerned as cytokines which counter and regulate the the production of IL-10. We suggest that these findings Th2 response by induction of tolerance and protect were the effects of both INC and antihistamine on from the development of allergic disorders.17,18 Rudin cytokine profiles of PBMCs of AR patients, because of et al.19 indicated that in vitro stimulation of PBMCs baseline effects of them and our assessment of patients with allergen showed an increased expression of the Th2-associated cytokines (IL-4, IL-5, IL-9, and IL-13), Altogether, it could be suggested that both natural whereas recent study by Heaton et al.20 showed that Ch.a and rCh.a induce IL-4 as a main Th2-associated PBMCs from both normal and allergic patients to house cytokine. Also, INC and antihistamine due to their anti- dust mites secreted comparable IFN-γ production after inflammatory effects can significantly inhibit the exposure to a mite extract. Our data are in agreement with both studies indicating that PBMCs from patients with AR produced higher IL-4 production. One goal of INCs and antihistamine drugs Previous studies have shown that INC treatment prescribed are to control allergic symptoms. In any results in ablation of the late phase allergic reaction, case, the immunomodulatory effects of these drugs are possibly due to the selective inhibition of cytokines important. Previous studies and our study have shown such as IL-4 and IL-5 expression.21-23 Ghaffar et al.24 that these drugs are able to modulate allergic reaction; found that 6-weeks topical gluccocorticosteroid therefore INCs and antihistamine as first-line for treatment in subjects with allergic rhinitis induced a treatment of patients are suggested. The assessment of significant reduction in the number of IL-13-expressing cytokine profiles of PBMCs of AR patients several 228/ IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY Effect of Intranasal Corticosteroid and Oral Antihistamine Treatment on Allergic Rhinitis times during the course of prescribed drugs is limitation budesonide on allergen challenge-induced nasal mucosal of this study. The results obtained in this experimental fluid levels of granulocyte-macrophage colony- study could be used for further investigations to explore stimulating factor and interleukin-5. Am J Respir Crit the involvement of cytokines effects and potency of INC and antihistamine compounds in protection of 10. Sim TC, Reece LM, Hilsmeier KA, Grant JA, Alam R. Secretion of chemokines and other cytokines in allergen- induced nasal responses: inhibition by topical steroid ACKNOWLEDGEMENTS
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