ORIGINAL ARTICLE

Role of BAFF in pediatric patients with allergic rhinitisduring sublingual immunotherapy

Renzhong Luo & Wenlong Liu & Jie Wang & Yanqiu Chen &

Changzhi Sun & Lifeng Zhou & Yan Li & Li Deng

Received: 27 November 2013 /Revised: 9 February 2014 /Accepted: 16 February 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract Sublingual immunotherapy (SLIT) is the only ther-apeutic option for allergic rhinitis (AR) that modifies the im-munological process to an allergen, rather than treating symp-toms simply. However, its regulatory mechanisms are largelyunknown. B-cell-activating factor of the TNF family (BAFF)plays very important roles in the development, differentiation,and proliferation of B cells and T cells. The aim of this studywas to identify the role of BAFF during SLIT in pediatricpatients with AR. Seventy-two house dust mite (HDM)-sensi-tized pediatric patients with AR were enrolled in this study.Thirty-six pediatric patients received HDM allergen extract forSLIT and 36 pediatric patients received placebo. Serum andnasal aspirate of different time points during treatment was

collected and used for enzyme-linked immunosorbent assay(ELISA) of BAFF and related cytokines, respectively. Periph-eral blood mononuclear cells were collected and stimulated byHDM allergen with or without rhBAFF after 12 months oftreatment. Our results showed that the expression of BAFFprotein decreased during the SLIT treatment compared withthat in the placebo group after 6 months of therapy, and thistrend lasted for 12 months. The decreased BAFF expressionwas positively related to Th2 cytokines and increased IL-10expression. BAFF was also related to local production of IgA.In vitro experiments showed that BAFF can promote Th2cytokines and inhibit IL-10 expression by PBMCs.Conclusion:During SLIT, BAFF expression was decreased and related tolow Th2 cytokine expression and enhanced IL-10 expression.Besides, BAFF may contribute to local production of IgA. Ourresults suggested that BAFF may be an important biomarkerduring SLIT. Authors’ summary. Sublingual immunotherapy(SLIT) is the only therapeutic option for allergic rhinitis (AR)that modifies the immunological process to an allergen, ratherthan simply treating symptoms. However, its regulatory mech-anisms are largely unknown. B-cell-activating factor of theTNF family (BAFF) plays very important roles in the develop-ment, differentiation, and proliferation of B cells and T cells.Our results showed that during SLIT, BAFF expression wasdecreased and related to low Th2 cytokine expression andenhanced IL-10 expression. Besides, BAFF may contribute tolocal production of IgA. Our results suggested that BAFF maybe an important biomarker during SLIT.

Keywords Allergic rhinitis . Sublingual immunotherapy .

B-cell-activating factor

AbbreviationsAR Allergic rhinitisBAFF-R BAFF receptorBAFF B-cell-activating factor of the TNF family

Communicated by David Nadal

Renzhong Luo, Wenlong Liu, and Jie Wang contribute equally to thisstudy.

R. Luo :W. Liu (*) : J. Wang :Y. Chen : C. Sun : L. Zhou :Y. Li :L. Deng (*)Department of Otolaryngology, Guangzhou Women and Children’sMedical Center, Guangzhou Medical College, No. 9 Jinsui Road,Guang Zhou 510623, Chinae-mail: lwl20103@163.come-mail: drdengli@126.com

R. Luoe-mail: 691285341@qq.com

J. Wange-mail: wangjie@163.com

Y. Chene-mail: frankcyq@163.com

C. Sune-mail: schzh@126.com

L. Zhoue-mail: zhoulifen@163.com

Y. Lie-mail: liya12@163.com

Eur J PediatrDOI 10.1007/s00431-014-2287-5

ELISA Enzyme-linked immunosorbent assayHDM House dust mitePBMCs Peripheral blood mononuclear cellsSPT Skin prick testSMS Symptom Medication ScoreTACI Transmembrane activator and CAML interactor

Introduction

Allergic rhinitis (AR) affects 30–40 % of the total populationworldwide, and its incidence is increasing [1, 23]. AR fre-quently coexists with asthma (most studies report a rate of 50–60 %) [10]. Sublingual immunotherapy (SLIT) is the onlytreatment that regulates the immunological process duringdevelopment of AR, rather than simply treating symptoms.SLIT affects the immune response by changing it from a Th2-predominant response toward a Th1 response accompanied bythe decreased infiltration of eosinophils and an increased IgGresponse, with raised IgG4 levels [8, 25]. However, the un-derlying mechanism in the process and potential biomarkersare still not fully characterized.

B-cell-activating factor, a member of the TNF family, playsimportant roles in the survival, proliferation, and maturationof B cells [9, 22]. In addition, BAFF is a costimulatory factorfor T cell activation and proliferation [17]. Although class-switch recombination is generally thought to be highly depen-dent on ligation of CD40 (on B cells) and CD40 ligand (onactivated T cells), it has been reported that BAFF also pro-motes T-cell-dependent immunoglobulin production as wellas CD40-independent and T-cell-independent immunoglobu-lin class switching and production [11, 16, 26]. BAFF canbind to three receptors that are selectively expressed on B cellsand plasma cells, including BAFF receptor (BAFF-R), trans-membrane activator and CAML interactor (TACI), and B cellmaturation antigen. BAFF can be produced by many types ofcells, such as monocytes, macrophages, dendritic cells, neu-trophils, salivary gland epithelial cells, and astrocytes8. A highlevel of serum BAFF exists in the patients with autoimmunediseases such as rheumatic arthritis, autoimmune diabetes,Sjogren’s syndrome, and multiple sclerosis [5, 28]. It wasreported that BAFF is probably very important in IgE-dependent allergic asthma, as B-cell-derived specific IgEserves as a mediator to trigger the allergic responses [15].

In the current study, we investigated whether BAFFmight be involved in the pathogenesis of SLIT. Wefound that serum and nasal aspirate from AR patientsduring SLIT had decreased levels of BAFF protein aswell as Th2 cytokines and IgA. These findings implythat downregulation of BAFF during SLIT may inhibitTh2 inflammation via induction of class-switch recom-bination and production of IgA by B cells in AR.

Methods

Patients

Seventy-two pediatric patients aged 5–16 years with aclinical history of monohouse dust mite (HDM)-inducedAR for at least 2 years were included. Thirty-six ofthem were enrolled in SLIT group, and the remainingpatients were included into the placebo group. Skinprick test (SPT) or specific immunoglobulin IgE (85.2±24.6 IU/mL) was performed to screen patients allergicto HDM. The cutoff value of the specific IgE is 3.5 IU/mL, and positive SPT was defined as mean whealdiameter ≥2 mm. No differences in specific IgE/SPTwheal size were found between the two groups. Thosewith chronic diseases (e.g., asthma, malnutrition, anatomic malformation of the respiratory system, chroniclung disease, heart disease, gastro-oesophageal refluxdisease, and cystic fibrosis) and those with a historyof chronic drug use (e.g., oral corticosteroids, antiepi-leptics, and immunosuppressives) were excluded fromthe study. The study was performed with the approvalof the local ethics committee and with the parent’swritten informed consent.

Sublingual immunotherapy

The HDM allergen extract (CHANLLERNGEN,Dermatophagoides farinae drops) for SLIT wasmanufactured by Wolwo Biotechnology PharmaceuticalCompany (Zhejiang, China) and used in the form of drops(no. 1, 1 mg/mL; no. 2, 10 mg/mL; no. 3, 100 mg/mL, and no.4, 333 mg/mL), and the biological activity of allergen extractswas determined by indirect ELISA assay, as approved by theChina Food and Drug Administration. According to the man-ufacturer’s instruction, the patients were asked to take increas-ing doses (from no. 1 to no. 3) during the first 3 weeks up-dosing phase and then were instructed to have 3 drops of no. 4solution once daily during the maintenance phase. Drops wereinstructed to be kept under the tongue for 2–3 min beforeswallowing. Patients in the placebo group received a diluentcontaining 50 % glycerol and 50 % saline buffer.

Clinical evaluation

Patient diary

The symptoms and medications were recorded using a patientdiary as described earlier [24]. All of the patients recordedtheir daily symptom score and drug requirement throughoutthe 12-month SLIT study.

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Symptom scores

The nose symptoms (runny nose, sneezing, and blockednose), eye symptoms (streaming and swelling and rednessand itching), and lung symptoms (breathlessness, cough,wheeze, and chest tightness) were scored by the children asfollows: 0=no symptoms, 1=slight symptoms, 2=moderatesymptoms, and 3=severe symptoms.

Medication scores

The daily medication score for each patient was calculated asthe sum of medication administered at a particular day:cetirizine tablets (10 mg)—2 points/tablet; cromolyn eyedrops (40 mg/mL)—1 point/drop; cromolyn nasal spray(5.2 mg/dose), terbutaline inhalation (0.25 mg/dose), andsalbutamol inhalation (0.2 mg/dose)—1 point/puff;budesonide inhalation (200 lg/dose); and fluticasone propio-nate inhalation (100 μg/dose)—4 points/puff and 80 pointsper course of prednisolone (5 mg/tablet).

The symptom and medication scores were recorded, and acombined Symptom Medication Score (SMS) was obtainedby adding together the two scores.

Blood and nasal aspirate sample preparation and analysis

Venous blood samples were collected into Vacuette tubes andcentrifuged at 3,000 g for 15 min at 4 °C. Serum samples werestored at−80 °C. These sampleswere used for EASIA and qPCRmeasurement. The whole blood cell counts were measured byLH-785 system (Beckman Coulter, Mervue, Galway, Ireland).Total serum IgE was measured by electrochemiluminescenceimmunoassay (ECLIA) method using an ELX-800 system.

Nasal aspirate was performed using saline warmed to37 °C. The process was performed according to methoddescribed elsewhere [14]. The samples were centrifuged toremove cellular debris, and aliquots of the supernatants werestored at −20 °C in Eppendorf tubes until analysis. Totalprotein concentrations were determined with Bio-Rad proteinassays according to Bradford.

Enzyme-linked immunosorbent assay (ELISA) for proteinexpression

ELISA kits were used for measuring serum BAFF, IL-4, IL-13,IL-5, IL-12, interferon (IFN)-α, IL-10, and transforming growthfactor (TGF)-β (R&D Systems, USA) according to the manu-facturer’s protocols. Total serum IgE and IgG levels were mea-sured by an ELX-800 system. The detection limits of the assayswere as follows: BAFF, 6.44 pg/mL; IL-4, 1.56 pg/mL; IL-13,93.8 pg/mL; IL-5, 7.8 pg/mL; IL-12, 2.5 pg/mL, IFN-α,12.5 pg/mL; IL-10, 3.9 pg/mL; and TGF-β, 15.4 pg/mL.

PBMC preparation

Peripheral blood mononuclear cells (PBMCs) were isolatedby Lymphoprep (Fresenius Kabi Norge AS, Oslo, Norway)density-gradient centrifugation from heparinized leukocyte-enriched buffy coats, which were obtained from the children1 year after the treatment. PBMCs were isolated by means ofdensity gradient centrifugation and cultured at 2×106/mL in24-well plates in culture medium: RPMI 1640 supplementedwith 5 % human AB serum, 5 mmol/L glutamine, and 13penicillin and streptomycin solutions (all from Invitrogen,except serum from Sigma-Aldrich). Stimulation was throughthe addition of allergen (HDM extract), with or without anti-BAFF or rhIL-BAFF.

Statistical analysis

Statistical analysis was performed using Mann–Whitney orWilcoxon signed rank test. The pretest Friedman test wasdone for multiple comparisons. The Spearman rank correla-tion test was used to analyze the correlation among the ex-pression of biomarkers and clinical stage. P<0.05 was con-sidered as significant difference.

Results

Demographic characteristics of study population and clinicaloutcome

This study was conducted with 72 patients, 36 of whomenrolled in SLIT group, with ages ranging between 61 and187 months (median age 110 months, 20 male patients) and36 of whom enrolled in SLIT group with ages ranging be-tween 65 and 192 months (mean age 115 months, 17 malepatients).The SLIT treatment was effective, and the SMSscores decreased significantly compared with the placebogroup (Table 1). Besides, the nasal score changed from 2.3(1.1, 3.0) to 0.8 (0.4, 1.3), and the lung score changed from 2.0(0.8, 2.6) to 0.9 (0.6, 1.5). Both organs were influenced in asimilar way.

Decreased serum and nasal BAFF protein levels during SLITtreatment

The serum and nasal BAFF protein expressions during SLITtreatment were significantly lower than those in the placebogroup, especially after 1-year treatment (Fig. 1a, b). Ourresults also showed decreased protein expression of eosino-phil cationic protein (ECP), IgA, and IgE 1 year after SLITtreatment (Fig. 1c–e). Correlation analysis showed that after1-year SLIT treatment, decreased BAFF expression was

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positively related to IgE, IgA, and ECP expressions, respec-tively (Fig. 2a–c).

Th1/Th2 cytokine expression during SLITand their relationship with BAFF expression

ELISA detection showed enhanced Th1 cytokine expres-sion and decreased Th2 cytokines after SLIT treatment.One year after SLIT treatment, IL-12 and IFN-α expres-sions increased significantly, while IL-4, IL-5, and IL-13 decreased significantly (Fig. 3a–e). However, thisTh1 polarization did not change significantly 6 monthsafter treatment. We also found that BAFF expressionwas negatively related to Th1 cytokines and positivelyrelated to Th2 cytokines (Fig. 4a–e).

Expression of protective protein in SLIT

ELISA detection showed that IL-10 expression increasedsignificantly 6 months after treatment, while TGF-β changed1 year after SLIT.We also found that IgG expression increasedduring SLIT as expected. We also found positive relationbetween BAFF and IL-10 as well as TGF-β expression(Fig. 5a–e). However, no relation was found between BAFFand IgG expression (data not shown).

Anti-BAFF alleviates Th2 inflammation of PBMCs inducedby HDM

After stimulation with HDM extract combined with anti-BAFF, we found significant downregulation of Th2cytokine (IL-4, IL-5, and IL-13) levels and upregulationof IL-10 in PBMCs (Fig. 6a–d), while Th1 cytokinelevels were not changed (data not shown). Yet, IgEand ECP decreased when the anti-BAFF was added inPBMCs (Fig. 6e–f).

Discussion

In the present study, the novel finding is that we found thatdecreased BAFF expression during SLIT treatment was cor-related with reduced Th2 cytokines and enhanced Th1 cyto-kines. In vitro study showed that BAFF may contribute toTh2/Th1/Treg regulation during SLIT.

SLIT is a useful treatment for AR, especially when therange of allergens is narrow. Successful SLIT is accompaniedby a reduction in Tcell and eosinophil recruitment in responseto allergen [2]. In parallel, there is a shift in the balance of Th1and Th2 cytokine expressions in the allergen-challenged site[6]. However, studies on efficacy of SLIT in Asia, especiallyin China, were few and inconsistent [20, 21, 27]. Our resultsshowed that SMS scores decreased significantly after 6-monthSLIT and that the improvement lasted for 1 year. Besides, nolife-threatening adverse events were reported. The similartrend in the improvement of nasal and lung symptoms maybe explained by the theory of “one airway, one disease”. Ourfindings showed that SLIT is effective and safe for Chinesepediatric patients.

BAFF is best known for its role in the survival and matu-ration of B cells and plays important roles in autoimmunediseases. As for allergic diseases, Kang et al. reported thatserum BAFF level was increased in patients with asthma andthat disease severity correlated with serum BAFF level [13].They suggested that BAFF might be an effective additionalindicator to monitor the severity of asthma symptoms. Inaddition, scavenging of BAFF was found to improve asthmat-ic symptoms. Besides, new evidence from BAFF transgenicmice indicates that BAFF induces the differentiation of IL-10-producing B cells [7, 18, 19]. Thus, we postulated that BAFFmay play important roles in SLIT treatment. Our resultsshowed that BAFF protein expression decreased 3 monthsafter the SLIT treatment without statistical significance, andthis downregulation becomes significant gradually and lastedfor 1 year after treatment. Interestingly, ECP, IgA, and IgEalso decreased as the same trend with BAFF. Consistent withour results, BAFF has been reported to be involved in

Table 1 Demographic characteristic of 72 AR children

SLIT group Placebo group

Number 36 36

Sex (male/female) 17:19 18:18

Age (months) 110 (61, 187) 115 (65, 192)

Endpoint versus baseline symptoms 2.7 (1.5, 4.2)* versus 6.7 (4.2, 9.0) 7.0 (4.5, 9.2) versus 6.8 (4.1, 8.9)

Endpoint versus baseline medication 4.9 (2.7, 7.1)* versus 7.3 (3.7, 10.3) 7.5 (3.8–10.6) versus 7.1 (3.9, 11)

Endpoint versus baseline SMS scores 7.9 (3.5, 12.2)* versus 12.9 (8.5, 15.6) 11.8 (7.9, 14.8) versus 12.5 (7.2, 13.9)

All data were presented as medians (highest and lowest value)

*P<0.05 compared with the placebo group

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eosinophil infiltration [12], IgE production [4], class-switchrecombination, and IgA production [3]. Taken together, ourfinding suggested that BAFFwas closely related to the clinicalefficacy of SLIT.

One important effect of SLIT is the balancing of Th1/Th2/Treg. Thus, we further investigated Th1/Th2/Treg cytokinechanges during the treatment; as we expected, SLIT treatmentcaused a shift from Th2 to Th1 inflammation and enhancedTreg cytokine expression. Interestingly, our results showedthat Th2 cytokines decreased after 6 months of SLIT, whileTh1 cytokines enhanced 1 year after SLIT. This lagged Th1polarization suggests that immunological changes of AR afterSLIT are far behind the improvement of symptoms. IL-10 andTGF-β are important protective cytokines as described in

literature. IL-10 inhibits synthesis of both Th1 and Th2 in-flammatory cytokines. TGF-β is believed to be important inthe regulation of the immune system. Our data showed thatIL-10 increased at the early stage of treatment, while TGF-βincreased at the late stage of treatment. Besides, the positiverelation between BAFF and IL-10 and TGF-β suggested thatBAFF was involved in IL-10 and TGF-β regulations. Inter-estingly, our study showed that Th2 cytokines were morestrongly correlated with BAFF compared with Th1/Treg cy-tokines, suggesting that BAFF was closely related to Th2inflammation and had a relative weak regulation on Th1reaction. Furthermore, we stimulated PBMCs with allergencombined with anti-BAFF and found significant downregula-tion of Th2 cytokine levels and upregulation of IL-10 in

Fig. 1 Serum and nasal BAFF protein expressions during SLIT weresignificantly lower than those in the placebo group (a, b). Decreasedprotein expression of ECP and IgA and IgE during SLIT than those in the

placebo group (c–e). *P<0.05, comparison between the two groups. Thecircle represents the placebo group and the square represents the SLITgroup

Fig. 2 Decreased BAFF expression was positively related to IgE, IgA, and ECP expressions (a–c). *P<0.05, comparison between the two groups

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PBMCs. Besides, IgE and ECP decreased when anti-BAFFwas added in PBMCs, while IL-10 increased significantly.

These results suggest that BAFF had a direct regulatory effecton Th1/Th2 balance in SLIT.

Fig. 3 Increased Th1 cytokines (IL-12 and IFN-α) and decreased Th2 cytokines (IL-4, IL-5, and IL-13) after SLIT (a–e). *P<0.05, comparison betweenthe two groups. The circle represents the placebo group and the square represents SLIT group

Fig. 4 BAFF expressionwas negatively related to Th1 cytokines and positively related to Th2 cytokines (a–e). *P<0.05, comparison between the two groups

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Collectively, our finding showed that during SLIT, BAFFexpression was decreased, and this low BAFF expression

lasted for 1 year after treatment. Decreased BAFF expres-sion was related to low Th2 cytokine expression and

Fig. 5 Increased IL-10, TGF-β, and IgG after SLIT (a–c). BAFF expression was positively related to IL-10 and TGF-β (d, e). *P<0.05, comparisonbetween two the groups. The circle represents the placebo group and the square represents the SLIT group

Fig. 6 After stimulation with HDM extract combined with or withoutanti-BAFF, significant downregulation of Th2 cytokine (IL-4, IL-5, andIL-13) levels and upregulation of IL-10 in PBMCs (a–d). IgE and ECPdecreased when anti-BAFF was added in PBMCs (e–f). *P<0.05,

comparison between the two groups (Group 1 100 ng/mL HDM, Group2 100 ng/mL HDM+100 ng/mL rhBAFF, and Group 3 100 ng/mLHDM+100 ng/mL anti-BAFF)

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enhanced IL-10 expression. BAFF may be used as abiomarker for SLIT treatment.

Conflict of interest The authors declare that they have no conflict ofinterest.

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