Clinical immunology
Specific sublingual immunotherapy decreases spontaneous and stimulated basophils degranulation. Preliminary studies

Introduction
Basophils and mast cells are the main effector cells responsible for IgE-dependent reactions. High affinity receptors for IgE immunoglobulin (FceRI) are present on the surface of those cells. When receptors bind IgE cross-linked with allergens, intracellular chains cross over and the cell degranulate. A large panel of mediators including cytokines and histamine, is released into extracellular space due to degranulation process. In addition, changes in the expression of basophil surface’s molecules are observed: some antigens appear de novo (CD63, CD107a), and other increase their expression (CD13, CD203c). The CD203c antigen is one of the most important marker of basophil’s activation. This protein (ectonucleotide pyrophosphatase/phosphodiesterase 3, E-NPP3, PD-Ib, B10, gp130RB13-6) belongs to type I phosphodiesterase/nucleotide pyrophosphatase family [1]. Its physiological function is unknown. The CD203c was found exclusively on basophils and mast cells. Its expression increases after basophil’s degranulation and therefore is a reliable marker of basophil’s activation in IgE-dependent process. Resting basophils are characterized by low expression of this protein and the expression rapidly increases when activated [2, 3].
The only effective causative treatment of atopic diseases is immunotherapy [4]. Subcutaneous immunotherapy (SCIT) was first described in late 60’s of XX century [5]. The method is based on injection of allergens in increased concentration to desensitize effector cells against specific antigens [6]. Unfortunately SCIT dosage is associated with possibility of anaphylactic reactions to allergens and has to be delivered in hospital [4]. Sublingual immunotherapy (SLIT) is an alternative to subcutaneous route of delivery. There is a few information about acute systemic reactions after SLIT [7], this route of delivery is patient friendly (especially in childhood) and can be administered at home [4]. Clinical efficacy of SLIT was already proved, but there is still limited number of studies about its influence on basophils activation process.
The aim of the study was to evaluate idiopathic and stimulated CD203c expression on basophils before and after twelve months of specific sublingual immunotherapy.

Materials and Methods
Six individuals aged of 8 ± 2.4 years old suffering from atopic disease which was confirmed with skin prick test (positive to Dermatophagoides pteronyssinus allergens), shortlisted for specific immunotherapy, served as studied group. Neither of analyzed subjects was treated with antihistamine drugs or oral corticosteroids. 1 ml blood taken by venipuncture in tubes containing EDTA was collected. Tests were performed before and after twelve months of specific sublingual immunotherapy. Patients were taking Staloral 300 in concentration of 300 IR/ml as often as prescribed by physician. The experiments were approved by the Ethics Commission of Medical University of Warsaw. Blood was collected with parents approval.
Basophil’s activation tests
All tests were carried out within two hours from blood collection. Anticoagulated blood was used for complete blood count analysis. Residual sample (400 µl) was used for basophil’s activation test. CD203c induced expression was evaluated using the Allergenicity Kit (Beckman Coulter) according to the manufacturer’s instructions. Allergens (Stallergenes, France) used for the test were prepared in 1:500 dilution of concentration used for skin prick tests in phosphate buffered saline (PBS) [8].
Briefly, EDTA-anticoagulated peripheral blood aliquots (100 µl) stained with 20 µl of mixture of monoclonal antibodies (CRTH2-FITC, CD203-PE, CD3-PC7) and Activation solution (100 µl) were stimulated (37°C) for 15 min with 20 µl of optimal dilution of allergens; antibody directed against the high affinity IgE receptor (FceRI) (Beckman Coulter) was used as positive control and PBS as a negative control. After incubation the reaction was stopped with Stop solution. Erythrocytes were lysed with Lysing solution for 10 minutes in room temperature (RT). Suspension was centrifuged (5 min, 300 g) after lysing, washed with PBS, once more centrifuged and resuspended in 500 µl Fixative solution. Leukocytes were analyzed using a five-color flow cytometer (Cytomics FC500, Beckman Coulter). During acquisition basophils were selected as CD203c positive/CRTH2 high/CD3 negative population using FL1/FL2 and SS/FL5 dot plots. In negative control threshold for positivity was set at less than 5% of activated cells according to the literature data [1, 9]. In positive control sensitivity for IgE dependent reaction was verified. Threshold for positive reaction was settled at less than 15% of activated cells, according to literature data [1, 10] and investigators studies’ results.
Statistical analysis
Results are presented as a mean ± SD. Statistical analysis was performed using the Wilcoxon matched pair test. A p value of less than 0.05 was considered significant.

Results
The number of resting basophils with presence of CD203c antigen on cell surface was counted before allergen stimulation. The mean fluorescence of fluorochrome bound to CD203c was measured. In patients before specific immunotherapy mean fluorescence of spontaneously activated basophils was 7.85 ± 5.17 MCF units, whereas after the year of immunotherapy the result was 4.22 ± 1.14 MCF, p < 0.05 (Fig. 1).
Average change is presented in Fig. 2. The D2 regions contain less than 5% of cells, as it is established for negative control.
The number of basophils with CD203c presence was evaluated to assess patients sensitivity to D. pteronyssinus antigens stimulation. Presence of CD203c was detected on 42.26 ± 5.12% basophils after incubation with specific allergens, whereas after one year of sublingual immunotherapy presence of CD203c was detected on 25.52 ± 12.8% cells,
p < 0.05.
The change in the number of CD203c positive basophils stimulated with D. pteronyssinus in the samples from selected patient before and after twelve month immunotherapy is presented in Fig. 4. Before SLIT 54.21% of basophils were activated after allergen stimulation, whereas after immunotherapy only 6.18% of basophils degranulated under the influence of specific antigen’s extract.

Discussion
There is an increased interest in SLIT all over the world. Sublingual immunotherapy, which can be administered at home and carry a very low risk of anaphylactic response as a side effect, is a promising alternative for subcutaneous immunotherapy [11]. It is postulated that SLIT is attractive option, especially for children. Its clinical efficacy has been reported recently. Improved quality of life, reduced drug intake (antihistamine and inhalant steroids) and decreased incidence of allergic reaction after SLIT was widely described [12]. Rodriguez-Perez et al. have summarized results of research in which effectiveness of 18 months SLIT is evident. Nevertheless this type of immunotherapy was effective only in grass pollen allergy, but not house dust mite [13, 14]. Immunological effect of SLIT are evaluated rarely, especially in children’s population.
Specific immunotherapy is proved to decrease total and specific IgE concentrations. While the specific IgG4 concentration increases, even up to 30-fold after one year of desensitization. It has to be pointed that only high dose SLIT caused satisfactory effect [15, 16]. Variety of immunological markers have been analyzed to prove efficacy of specific immunotherapy. Investigators tested serum concentration of ECP (eosinophil cationic protein), the marker of eosinophils degranulation, to evaluate decrease of eosinophils activity. The ECP concentrations were decreased after 6, 12 and 24 month of SLIT. Moreover eosinophil’s binding to ICAM-1 particles was declined, which may explain decrease of number of asthmatic incidences. Reduction of IL-13, cytokine, released from Th2 lymphocytes, was observed as well as increased Th1/Th2 ratio. It indicates that long lasting SLIT can influence immunoregulation [5]. On the other hand short-term SLIT, in spite of clinical efficacy, neither restores immunological balance in released cytokines profile nor alters levels of allergen-specific immunoglobulins E [17].
The goal of the present paper was to assess one year sublingual immunotherapy effect on CD203c basophil’s activation marker. The paper is very original as no comparative data were found. Although it was described that histamine release (secondary to basophils degranulation) is reduced in patients after SCIT [5], no publication reporting histamine release changes after SLIT treatment was found.
Our findings are in the line with observations that one year long SLIT is clinically effective [18]. Reduction of resting and post-stimulatory basophils activation level may be related to clinical efficacy. Usually patients after SLIT more rarely take antihistamine drugs or oral corticosteroids which suppress allergic response and as the result evaluation of their quality of life is raised comparing to placebo group [12]. Further analysis of the studied group will be performed.
The mechanism of sublingual immunotherapy is largely unknown. The allergens from the vaccin cross mucosal barrier and are potentially directed to gut-associated lymphoid tissue (GALT) where are exposed to immunological system [5]. The contact leads to development of regulatory T cells secreting TGF-b [11,19]. Moreover the exposure of allergens to immunological system causes increased production of specific IgG4. G4 immunoglobulin prevents allergen’s binding to IgE, which is the direct mechanism of basophils degranulation’s decrease. Immunotherapy influences also on B-cell production of IgA. Increase of IgA concentration enhances mucosal barrier and prevents allergen’s penetration. It is also postulated that SLIT can sensitize Th2 cells to apoptosis, which might be the mechanism of restoring Th1/Th2 balance [20].
Our paper sheeds light into mechanisms of the clinical efficacy of sublingual immunotherapy to D. pteronyssinus allergens on cellular level. As it is postulated that SLIT modifies immune response in different way than SCIT [21], further investigations are needed to assess influence of SLIT on immunological system.

References
1. Boumiza R, Monneret G, Forissier M-F et al. (2003): Marked improvement of the basophil activation test by detecting CD203c instead of CD63. Clin Exp Allergy 33: 259-265.
2. Ebo DG, Sainte-Laudy J, Bridts CH et al. (2006): Flow-assisted allergy diagnosis: current applications and future perspectives. Allergy 61: 1028-1039.
3. Triggiani M, Marone G (2006): Basophil’s secrets revealed by flow cytometry. Allergy 61: 1025-1027.
4. Cox L (2007): Sublingual immunotherapy in pediatric allergic rhinitis and asthma: efficacy, safety, and practical considerations. Curr Allergy Asthma Rep 7: 410-420.
5. Leatherman BD, Owen S, Parker M et al. (2007): Sublingual Immunotherapy: Past, present, paradigm for the future? A review of the literature. Otolaryngol Head Neck Surg 136 (3 Suppl): S1-20.
6. Larenas-Linnemann D (2008): Subcutaneous and sublingual immunotherapy in children: complete update on controversies, dosing, and efficacy. Curr Allergy Asthma Rep 8: 465-474.
7. Rodríguez-Pérez N, Ambriz-Moreno Mde J, Canonica GW, Penagos M (2008): Frequency of acute systemic reactions in patients with allergic rhinitis and asthma treated with sublingual immunotherapy. Ann Allergy Asthma Immunol 101: 304-310.
8. Potapinska O, Górska E, Zawadzka-Krajewska A et al. (2009): The usefulness of CD203c expression measurement on basophils after activation with grass pollen and Dermatophagoides pteronyssinus antigens. Preliminary study. Pneumonol Allergol Pol (in press).
9. Sudheer PS, Hall JE, Read GF et al. (2005): Flow cytometric investigation of per-anaesthetic anaphylaxis using CD63 and CD203c. Anaesthesia 60: 251-256.
10. Eberlein-Konig B, Varga R, Mempel M et al. (2006): Comparison of basophil activation tests using CD63 or CD203c expression in patients with insect venom allergy. Allergy 61: 1084-1085.
11. Cox LS, Larenas Linnemann D, Nolte H et al. (2006): Sublingual immunotherapy: a comprehensive review. J Allergy Clin Immunol 117: 1021-1035.
12. Passalacqua G, Pasquali M, Ariano R et al. (2006): Randomized double-blind controlled study with sublingual carbamylated allergoid immunotherapy in mild rhinitis due to mites. Allergy 61 :849-854.
13. Rodríguez-Pérez N, Penagos M, Portnoy JM (2008): New types of immunotherapy in children. Curr Allergy Asthma Rep 8(6):484-92.
14. Larenas-Linnemann D.(2009): Sublingual immunotherapy in children: complete and updated review supporting evidence of effect. Curr Opin Allergy Clin Immunol 9: 168-176.
15. Rossi RE, Monasterolo G, Coco G et al. (2007): Evaluation of serum IgG4 antibodies specific to grass pollen allergen components in the follow up of allergic patients undergoing subcutaneous and sublingual immunotherapy. Vaccine 25: 957-964.
16. Rolinck-Werninghaus C, Kopp M, Liebke C et al. (2005): Lack of Detectable Alterations in Immune Responses during Sublingual Immunotherapy in Children with Seasonal Allergic Rhinoconjunctivitis to Grass Pollen Int Arch Allergy Immunol 136: 134-141.
17. Dehlink E, Eiwegger T, Gerstmayr M et al. (2006): Absence of systemic immunologic changes during dose build-up phase and early maintenance period in effective specific sublingual immunotherapy in children Clin Exp Allergy 36: 134-141.
18. Ott H, Sieber J, Brehler R et al. (2009): Efficacy of grass pollen sublingual immunotherapy for three consecutive seasons
and after cessation of treatment: the ECRIT study. Allergy 64: 179-186.
19. Savolainen J, Jacobsen L, Valovirta E (2006): Sublingual immunotherapy in children modulates allergen-induced in vitro expression of cytokine mRNA in PBMC Allergy 61: 1184-1190.
20. Tsai YG, Chien JW, Chen WL et al. (2005): Induced apoptosis of TH2 lymphocytes in asthmatic children treated with Dermatophagoides pteronyssinus immunotherapy. Pediatr Allergy Immunol 16: 602-608.
21. Antúnez C, Mayorga C, Corzo JL et al. (2008): Two year follow-up of immunological response in mite-allergic children treated with sublingual immunotherapy. Comparison with subcutaneous administration. Pediatr Allergy Immunol 19: 210-218.