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-Dependent MechanismβIFN-Cell-Activating Factor of TNF Family by an Airway Epithelial Cells Produce B

Matsumoto and Robert P. SchleimerAtsushi Kato, Ai Q. Truong-Tran, Alan L. Scott, Kenji

http://www.jimmunol.org/content/177/10/7164doi: 10.4049/jimmunol.177.10.7164

2006; 177:7164-7172; ;J Immunol 

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Airway Epithelial Cells Produce B Cell-Activating Factorof TNF Family by an IFN-�-Dependent Mechanism1

Atsushi Kato,* Ai Q. Truong-Tran,* Alan L. Scott,† Kenji Matsumoto,‡ andRobert P. Schleimer2*

Activation of B cells in the airways is now believed to be of great importance in immunity to pathogens, and it participates in thepathogenesis of airway diseases. However, little is known about the mechanisms of local activation of B cells in airway mucosa.We investigated the expression of members of the B cell-activating TNF superfamily (B cell-activating factor of TNF family(BAFF) and a proliferation-inducing ligand (APRIL)) in resting and TLR ligand-treated BEAS-2B cells and primary humanbronchial epithelial cells (PBEC). In unstimulated cells, expression of BAFF and APRIL was minimal. However, BAFF mRNA wassignificantly up-regulated by TLR3 ligand (dsRNA), but not by other TLR ligands, in both BEAS-2B cells (376-fold) and PBEC(224-fold). APRIL mRNA was up-regulated by dsRNA in PBEC (7-fold), but not in BEAS-2B cells. Membrane-bound BAFFprotein was detectable after stimulation with dsRNA. Soluble BAFF protein was also induced by dsRNA (>200 pg/ml). Thebiological activity of the epithelial cell-produced BAFF was verified using a B cell survival assay. BAFF was also strongly inducedby IFN-�, a cytokine induced by dsRNA. Induction of BAFF by dsRNA was dependent upon protein synthesis and IFN-��receptor-JAK-STAT signaling, as indicated by studies with cycloheximide, the JAK inhibitor I, and small interfering RNA againstSTAT1 and IFN-�� receptor 2. These results suggest that BAFF is induced by dsRNA in airway epithelial cells and that theresponse results via an autocrine pathway involving IFN-�. The production of BAFF and APRIL by epithelial cells may contributeto local accumulation, activation, class switch recombination, and Ig synthesis by B cells in the airways. The Journal of Immu-nology, 2006, 177: 7164–7172.

A irway epithelial cells form a complex physical barrierthat defends against harmful substances and microbialpathogens. Airway epithelial cells also function in the

regulation of immune responses through production of cytokinesand chemokines and via interactions with cells of the immunesystem. Epithelial activation results from direct exposure of epi-thelial cells to injurious material, exposure to pathogen-associatedmolecular patterns (PAMPs)3 that trigger TLRs, or exposure toactivating cytokines derived from tissue cells or infiltrating leuko-cytes (1–6). Activation of epithelial cells can result in immediatehost defense responses, as they are induced to produce host de-fense molecules. In addition, prolonged or robust epithelial acti-vation can result in the release of proinflammatory cytokines andchemokines that attract inflammatory cells. Activation of epithe-

lium and production of cytokines and chemokines have been ofparticular interest in allergic conditions such as asthma and allergicrhinitis (7, 8). It has been studied widely because these mediatorsinfluence the activity of inflammatory cells such as eosinophils, Tcells, B cells, and mast cells, which are the characteristic infiltrat-ing cells in these disorders (7, 8).

The production of IgA and IgE from B cells has been known tobe critical in allergic diseases. Diaz-Sanchez et al. (9) demon-strated local IgE production in the upper airways of human sub-jects. Recent evidence has demonstrated that extensive Ig classswitch recombination (CSR) and IgE production occur in the nasalmucosa of patients with allergic rhinitis and in the bronchial mu-cosa of asthmatics (10, 11). In addition, activated IgA- and IgE-expressing B cells have been found in the airway mucosa of pa-tients with these diseases. Due to the recognized importance of IgEin asthma and rhinitis, the local activation of B cells in the airwayhas been proposed to be an important event in the pathogenesis ofallergic airway diseases.

CSR is a process by which B cells shift from expression of theIgM H chain C region (C�) to another C region to make a newisotype of Ab (e.g., switching from IgM to IgA or IgE). Ags elicitCSR in germinal center B cells through interactions between CD40on B cells and the TNF ligand superfamily (TNFSF) memberCD40L (also known as CD154 and TNFSF5) on activating CD4�

T cells (12, 13). Although CSR is generally thought to be highlydependent on CD40, it is also reported that viral and bacterialproducts can induce Ig production in the absence of T cells orCD40-dependent signaling (14–17).

A proliferation-inducing ligand (APRIL; also known asTNFSF13) and B cell-activating factor of the TNF family (BAFF;also known as BLyS, TNFSF13B, TALL-1, and THANK) are re-cently identified members of the TNFSF that play important rolesin B cell maturation and function (18–21). BAFF promotes B cell

*Allergy-Immunology Division, Northwestern University Feinberg School of Medi-cine, Chicago, IL 60611; †Department of Molecular Microbiology and Immunology,Johns Hopkins University Bloomberg School of Public Health, Baltimore, MD21205; and ‡Department of Allergy and Immunology, National Research Institute forChild Health and Development, Tokyo, Japan

Received for publication May 18, 2006. Accepted for publication August 22, 2006.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by National Institutes of Health Grants R01HL068546 and R01 HL078860 and by a grant from the Ernest S. Bazley Trust.2 Address correspondence and reprint requests to Dr. Robert P. Schleimer, Allergy-Immunology Division, Northwestern University Feinberg School of Medicine, 240East Huron, Chicago, IL 60611. E-mail address: rpschleimer@northwestern.edu3 Abbreviations used in this paper: PAMP, pathogen-associated molecular pattern;APRIL, a proliferation-inducing ligand; BAFF, B cell-activating factor of TNF fam-ily; BAFF-R, BAFF receptor; BCMA, B cell maturation Ag; CSR, class switch re-combination; FP, fluticasone propionate; IFNAR, IFN-�� receptor; JAKI, JAKinhibitor I; mBAFF, membrane-bound BAFF; PBEC, primary human bronchial epithe-lial cell; PGN, peptidoglycan; sBAFF, soluble BAFF; siRNA, small interfering RNA;TACI, transmembrane activator and CAML interactor; TNFSF, TNF ligand superfamily;Tyk2, tyrosine kinase 2.

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survival and proliferation (21–23). BAFF and APRIL also promoteCD40-independent, T cell-independent CSR and Ig production(15, 24). BAFF binds to three receptors that are selectively ex-pressed on B cells, including transmembrane activator and CAMLinteractor (TACI), B cell maturation Ag (BCMA), and BAFF re-ceptor (BAFF-R) (21–23). APRIL also binds to TACI and BCMA,but not BAFF-R, which only binds BAFF (21–23). BAFF-R is apotent regulator of mature B cell survival (25, 26). In contrast,TACI has been considered to suppress B cell proliferation andsurvival. BCMA does not appear to be important for B cell ho-meostasis, but is required for optimal survival of plasma cells.BAFF-R and TACI are also able to signal class switching to IgGand IgE, but the class switch to IgA is regulated by TACI only(24). It has been reported that elevated amounts of BAFF can befound in the serum of autoimmune patients, such as those withsystemic lupus erythematosus, rheumatoid arthritis, and Sjogren’ssyndrome, and the potential therapeutic efficacy of Abs againstBAFF has been investigated (22, 27, 28). BAFF transgenic micehave high levels of total Igs, rheumatoid factors, and circulatingimmune complexes in their serum, whereas animals or humanslacking BAFF or BAFF-Rs have minimal B cells and Ig responses(29, 30). Thus, it has been suggested that BAFF plays an importantrole in both normal immune responses and autoimmune diseasepathogenesis. In contrast to autoimmune disease, little is knownabout the role of BAFF and APRIL in inflammatory diseases of theairways.

Functional TLR family members are known to be expressed onairway epithelial cells (6, 31–34). In the present study, we inves-tigated whether members of the B cell-activating TNFSFs,CD40L, APRIL, and BAFF were expressed or induced by TLRligands in airway epithelial cells. We discovered that BAFF wasstrongly and significantly induced by the TLR3 ligand dsRNA andthat expression of BAFF is critically regulated by TLR3- and IFN-�-dependent signaling in airway epithelial cells. These findingsraise the possibility of an important interaction between airwayepithelial cells and B cells and lead to the hypothesis that theproduction of BAFF by epithelial cells may contribute to localaccumulation, activation, CSR, and Ig synthesis by B cells in theairway.

Materials and MethodsReagents

Zymosan A from Saccharomyces cerevisiae, LPS from Escherichia coli,serotype 0111:B4, and fluticasone propionate (FP) were purchased fromSigma-Aldrich. Poly(I:C) (dsRNA) (Amersham Biosciences), peptidogly-can (PGN) from Staphylococcus aureus (Fluka), flagellin isolated fromSalmonella typhimurium strain 14,028 (Apotech), and R-848 (InvivoGen)were purchased from the indicated sources. CpG oligodeoxynucleotide2216 (CpG-A; 5�-ggGGGACGATCGTCgggggG-3�, small letters, phos-phorothioate linkage; capital letters, phosphodiester linkage 3� of the base)was synthesized at Sigma Genosys. Recombinant human IL-1�, IL-4, IL-6,IL-10, IL-12, IL-15, TNF-�, IFN-�, IFN-�, G-CSF, GM-CSF, BAFF, andthe IgG1 Fc fusion proteins of BAFF-R (BAFF-R:Fc) and TACI (TACI:Fc) were purchased from R&D Systems. The small interfering RNAs (siR-NAs) against STAT1 (Hs_STAT1_6 HP validated siRNA; SI02662324),IFN-�� receptor 2 (IFNAR2) (Hs_IFNAR2_2 HP siRNA; SI00017500),and the no effect control were obtained from Qiagen.

Cell culture, treatments, and transfection

The adeno 12 SV40-transformed human bronchial epithelial cell line,BEAS-2B, was a gift from C. Harris (National Cancer Institute, Bethesda,MD) (35) and cultured in DMEM/F12 (Invitrogen Life Technologies) sup-plemented with 5% heat-inactivated FBS (Invitrogen Life Technologies), 2mM L-glutamine, 100 U/ml penicillin, and 100 �g/ml streptomycin (In-vitrogen Life Technologies). Human primary bronchial epithelial cells(PBEC) were obtained, as described previously (36). PBEC were main-tained in serum-free LHC-9 medium (BioSource International). PBECwere plated in 12- or 24-well culture plates coated with collagen (Vitrogen;

Collagen Biomaterials). Before stimulation, PBEC were cultured in serum-free LHC-8 medium without hydrocortisone (BioSource International) forat least 2 days. Either type of airway epithelial cells was stimulated withone of the following at the indicated concentration: 10 �g/ml PGN, 100�g/ml zymosan, 25 �g/ml dsRNA, 1 �g/ml LPS, 10 ng/ml flagellin, 1�g/ml R-848, and 3 �g/ml CpG-A for 18 h; or 100 ng/ml IL-1�, 100 ng/mlIL-4, 100 ng/ml IL-6, 100 ng/ml IL-10, 100 ng/ml IL-12, 100 ng/ml IL-15,100 ng/ml TNF-�, 1000 U/ml IFN-�, 100 ng/ml IFN-�, 100 ng/ml TGF-�,100 ng/ml G-CSF, and 100 ng/ml GM-CSF for 6 h.

BEAS-2B cells were seeded at low density (5 � 104 cells/well) over-night in DMEM/F12 supplemented with 5% heat-inactivated FBS. At 30–50% confluence, cells were transfected with siRNA against STAT1, IF-NAR2, or control RNA at 5 or 20 nM using HiPerFect transfection reagent(Qiagen), following the manufacturer’s instruction. The transfected cellswere further grown for 72 h, and then stimulated with 1000 U/ml IFN-�and 25 �g/ml dsRNA for 6 h.

Real-time PCR and RT-PCR

Total RNA was extracted using RNeasy (Qiagen) and was digested withDNase I (Qiagen), according to the manufacturer’s instructions. Single-strand cDNA was synthesized with SuperScript II reverse transcriptase(Invitrogen Life Technologies) and random primers. Real-time RT-PCRwas performed with a TaqMan method using an Applied Biosystems 7500Sequence Detection System (Applied Biosystems) in 20 �l reactions (10 �lof 2� TaqMan Master mix (Applied Biosystems), 400 nM each primer,and 200 nM TaqMan probe plus cDNA). Primer and probe sets for fourgenes, BAFF (sense, 5�-TCGATGTATTCAAAATATGCCTGAAA-3�;antisense, 5�-TGCAATGCCAGCTGAATAGC-3�; MGB-probe, 5�-CTACCCAATAATTCC-3�), APRIL (sense, 5�-AGAGCAGTGCTCACCCAAAAAC-3�; antisense, 5�-GCCACATCACCTCTGTCACATC-3�; MGB-probe, 5�-CACCTGGTTCCCATTAA-3�), CD40L (sense, 5�-CTCTATTATATCTATGCCCAAGTCACCTT-3�; antisense, 5�-GACTTTAGGCAGAGGCTGGCTA-3�; MGB-probe, 5�-CGAGTCAAGCTCC-3�), and GAPDH (sense, 5�-GAAGGTGAAGGTCGGAGTC-3�; antisense, 5�-GAAGATGGTGATGGGATTTC-3�; FAM/TAMRA-labeled probe, 5�-CAAGCTTCCCGTTCTCAGCC-3�) were synthesized at Applied Biosystems. Real-time PCR for IFN-� was performed with a SYBR Green I method, asdescribed previously (37). To determine the exact copy number of thetarget genes, quantified aliquots of purified PCR fragments of target geneswere serially diluted and used as standards in each experiment. Aliquots ofcDNA equivalent to 10 ng of total RNA (PCR for IFN-� was 125 ng) wereused for real-time PCR. The expression levels of mRNA were normalizedto the median expression of a housekeeping gene (GAPDH). ConventionalRT-PCR was performed using Platinum TaqDNA Polymerase in 50 �lreactions (45 �l Platinum PCR SuperMix (Invitrogen Life Technologies)and 200 nM each primer plus cDNA). BAFF primers for RT-PCR (sense,5�-GAAATAAGCGTGCCGTTCAGG-3�; antisense, 5�-TTAGATGTCCCATGGCGTAGGTC-3�) were synthesized at Applied Biosystems.

GeneChip expression analysis

GeneChip analysis was performed, as described previously (38). Gene ex-pression was measured using the GeneChip Human Genome U133 plus 2.0probe array (Affymetrix). Data analysis was performed with the GeneChipOperating Software (Affymetrix) and the GeneSpring software version 7.2(Agilent).

Flow cytometry

BEAS-2B cells were dispersed into a single-cell suspension by incubationwith Versene (Invitrogen Life Technologies) for 15 min. Cells were sus-pended in PBS containing 1.5% BSA and then incubated with either PE-conjugated mouse anti-human BAFF mAb (clone: 1D6, IgG1; eBio-science) or PE-conjugated mouse control IgG1 Ab (clone: MOPC-31C; BDPharmingen) for 30 min in the dark at 4°C. Cells were washed and resus-pended in PBS containing 0.1% BSA and immediately analyzed with a BDFACSArray Bioanalyzer (BD Biosciences). Flow cytometry data were an-alyzed using FlowJo software version 5.6.1 (Tree Star).

ELISA

The concentrations of BAFF protein in cell-free supernatants were mea-sured with a specific ELISA kit (R&D Systems). The detection limit forthis kit is 31.25 pg/ml.

B cell isolation

Human peripheral blood buffy coats were obtained from LifeSource BloodServices. Human PBMC were isolated by centrifugation on a Ficoll-sodium diatrizoate density gradient (d � 1.077 g/ml, Ficoll-Paque plus;

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Amersham Biosciences) from buffy coat, according to the manufacturer’sdirections. After lysing erythrocytes, B cells were purified using the MACSsystem (Miltenyi Biotec) with anti-CD19 MicroBeads (Miltenyi Biotec).The purity of the CD20-positive B cells prepared was always �99%.

MTT assay

For the analysis of the biological activity of secreted BAFF, BEAS-2Bcells were cultured in serum-free DMEM/F12 medium and then stimulatedwith 1000 U/ml IFN-� for 72 h. The supernatant was concentrated 150–200 times using Centriplus YM-10 (Millipore) and Microcon YM-10 (Mil-lipore). B cells were cultured at a density of 2 � 105 cells/100 �l/well ofa 96-well flat-bottom microtiter plate in RPMI 1640 (Invitrogen Life Tech-nologies) supplemented with 10% FBS, 100 U/ml penicillin, and 100�g/ml streptomycin. Cells were stimulated as indicated with 5 ng/mlBAFF, 10 ng/ml IL-4, 10 �g/ml mouse anti-human CD40 mAb (clone:5C3, IgG1; eBioscience), concentrated supernatant, or combinations of theabove for 5 days in the presence or absence of 5 �g/ml control IgG1, 5�g/ml BAFF-R:Fc, and 5 �g/ml TACI:Fc. To detect intracellular enzymeactivity, 10 �l of MTT reagent (American Type Culture Collection) wasadded for the last 4 h of incubation. After 4 h, 100 �l of detergent reagent(American Type Culture Collection) was added to all wells, and the platewas incubated in the dark overnight at room temperature. Absorbance at570 nm was measured using a microtiter plate reader.

Statistical analysis

All data are presented as the mean � SEM, unless otherwise noted. Dif-ferences between groups were analyzed using the paired Student’s t testand considered to be significant if p � 0.05.

ResultsEffect of TLR ligands on BAFF and APRIL expression in airwayepithelial cells

Growing evidence indicates that B cell proliferation and differen-tiation occur at mucosal surfaces. To test the possible role ofepithelial cells, we first set out to determine the spontaneous ex-

pression of mRNA for members of the B cell-activating TNFsuperfamily ligand such as CD40L, BAFF, and APRIL in unstimu-lated bronchial epithelial cells using GeneChip and real-time PCR.Neither CD40L (ID: 207892_at), BAFF (ID: 223501_at and223502_at), or APRIL (ID: 229326_at) was detected in unstimu-lated BEAS-2B cells using the GeneChip (data not shown). Usingreal-time PCR, mRNA for BAFF and APRIL were detected, butvery weakly expressed in both resting BEAS-2B cells and restingPBEC (Fig. 1, A and B). The level of mRNA for CD40L was belowthe detection limit of real-time PCR (data not shown).

To assess whether or not TLR ligands induce CD40L, BAFF,and APRIL expression, BEAS-2B cells and PBEC were treatedwith PGN (TLR2 ligand), zymosan (TLR2 ligand), dsRNA (TLR3ligand), LPS (TLR4 ligand), flagellin (TLR5 ligand), R-848(TLR7/8 ligand), and CpG-A (TLR9 ligand) for 18 h. mRNA forBAFF was significantly induced by stimulation with dsRNA inBEAS-2B cells (376-fold; n � 4; p � 0.05) (Fig. 1A). LPS induceda nonsignificant increase in BAFF expression in BEAS-2B cells(4-fold; n � 4; p � 0.068), whereas ligands for TLR 2, 5, 7/8, and9 had no effect. In contrast, mRNA for APRIL and CD40L werenot induced by any of the TLR ligands tested in BEAS-2B cells,even after stimulation up to 72 h (Fig. 1A and data not shown).Results were similar in PBEC, with the exception that BAFF wasnot induced by LPS, and APRIL was significantly induced only bydsRNA (7-fold; n � 4; p � 0.05) (Fig. 1B). BAFF mRNA wassignificantly up-regulated by dsRNA in PBEC (224-fold; n � 4;p � 0.05). We also examined the effect of TLR ligands on theinduction of BAFF and APRIL in human primary nasal epithelialcells. mRNA for BAFF and APRIL was also significantly inducedby dsRNA in human primary nasal epithelial cells, and these ef-fects were the same as those observed in PBEC (data not shown).

FIGURE 1. Effect of TLR ligands on the up-regulation of BAFF and APRIL in airway epithelial cells. BEAS-2B cells (A) or PBEC (B) were incubatedfor 18 h with 10 �g/ml PGN, 100 �g/ml zymosan, 25 �g/ml dsRNA, 1 �g/ml LPS, 10 ng/ml flagellin, 1 �g/ml R-848, or 3 �g/ml CpG-A, as indicated,and then mRNA was extracted and analyzed for BAFF and APRIL using real-time PCR. C, BEAS-2B cells or PBEC were incubated for 18 h with 25 �g/mldsRNA, and then expression of mRNA for full-length BAFF (246 bp), �BAFF (189 bp), and GAPDH (226 bp) was analyzed by conventional RT-PCR.D, BEAS-2B cells were incubated for 6 h with 0.25–25 �g/ml dsRNA, and then expression of mRNA for BAFF was analyzed by real-time PCR. E,BEAS-2B cells were incubated with 25 �g/ml dsRNA (circle) or vehicle control (square) for 0.5–18 h, and then expression of mRNA for BAFF wasanalyzed by real-time PCR. The copy number is expressed as the number of transcripts/ng total RNA. The results are shown as the mean � SEM of threeto five independent experiments. �, p � 0.05.

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Because the absolute levels of mRNA and relative increase in-duced by dsRNA were higher for BAFF than for APRIL, we havefocused our studies in BAFF. Recently, �BAFF, which has beenshown to be a natural negative regulator of BAFF, has been iden-tified. �BAFF is a spliced isoform of BAFF and it lacks exon 3containing 57 bp (39, 40). We examined whether the form ofBAFF induced by dsRNA was the active form or the inactive �form using conventional RT-PCR. Although the �BAFF isoformwas found to be expressed in the dsRNA-treated BEAS-2B andPBEC, it was only a minor isoform (Fig. 1C). To further examinethe details of the induction of mRNA for BAFF by dsRNA, wedetermined the concentration and the time dependence of the re-sponse in BEAS-2B cells. BAFF mRNA was induced by concen-trations of dsRNA as low as 0.25 �g/ml and with a time lag of 3h (Fig. 1, D and E).

BAFF exists as a type II membrane protein as well as a solubleprotein derived from the membrane-bound form by cleavage with aputative furin family protease (20, 21, 41). To confirm epithelial ex-pression of BAFF protein, we assayed the cell surface expressionusing flow cytometry and measured the concentration in the culturesupernatant using a specific ELISA. Low levels of membrane-boundBAFF (mBAFF) were detectable only after stimulation of BEAS-2Bcells with dsRNA (Fig. 2A). In contrast, significant levels of solubleBAFF (sBAFF) were detected in the supernatant after stimulationwith dsRNA. Appearance of sBAFF occurred in a time-dependentmanner in BEAS-2B cells (maximum at 72 h of culture: control, un-detectable; dsRNA treated, 228 � 8 pg/ml; n � 3; Fig. 2B). In PBEC,

sBAFF was also strongly induced by dsRNA (72-h culture: control,undetectable; dsRNA treated, 97 � 15 pg/ml; n � 3; Fig. 2C). Theseresults indicate that stimulation of airway epithelial cells with dsRNAinduces significant expression of both BAFF mRNA and BAFF pro-tein. To investigate the polarity of BAFF production, we used a trans-well system. After confluence, BEAS-2B cells were stimulated with25 �g/ml dsRNA for 48 h. The supernatant was concentrated to 100�l using Microcon YM-10. These showed that airway epithelial cellsproduced BAFF protein on both the apical and basolateral sides of themonolayer after stimulation with dsRNA (apical, 401 � 63 pg/ml;basolateral, 234 � 17 pg/ml; n � 4).

Effect of cytokines on BAFF expression in airway epithelial cells

Published studies indicate that BAFF is induced in monocytes,macrophages, dendritic cells, neutrophils, nurse-like cells, astro-cytes, and salivary gland epithelial cells by several cytokines, in-cluding IL-10, IFN-�, IFN-�, IFN-�, and G-CSF (15, 41–45). Wetherefore examined whether cytokines were able to induce BAFFexpression in airway epithelial cells. BEAS-2B cells and PBECwere incubated with IL-1�, IL-4, IL-6, IL-10, IL-12, IL-15,TNF-�, IFN-�, IFN-�, TGF-�, G-CSF, and GM-CSF for 6 h.mRNA for BAFF was significantly up-regulated by TNF-� (6-fold; n � 4; p � 0.05), IFN-� (485-fold; n � 4; p � 0.05), andIFN-� (26-fold; n � 4; p � 0.05), but not by the other cytokinestested in BEAS-2B cells (Fig. 3A). Similar results were obtained inPBEC, with the exception that TNF-� did not significantly induceBAFF (Fig. 3B). BAFF was induced by IFN-� (21-fold; n � 4;p � 0.05) and IFN-� (2-fold; n � 4; p � 0.05) in PBEC (Fig. 3B).

Role of IFN-� in dsRNA-induced BAFF expression

Among the cytokines tested, IFN-� induced the highest level ofBAFF expression. We therefore investigated further the potentialrole that IFN-� may play in the induction of mRNA for BAFF.BEAS-2B cells were incubated with different concentrations of

FIGURE 2. Detection of BAFF protein in airway epithelial cells. A, Rep-resentative histograms from flow cytometry demonstrate evidence of mBAFFon BEAS-2B cells. Cells were stained with mAb specific for BAFF (dark line)or an isotope control mAb (shaded histogram) after incubation with or without(untreated) 25 �g/ml dsRNA for 24 h. Detection of sBAFF protein by ELISAin the culture supernatant of BEAS-2B (B) and PBEC (C, 72 h) stimulated withdsRNA for the indicated time. Results shown are mean � SEM of three to fourindependent experiments. **, Not detectable. �, p � 0.05.

FIGURE 3. Effect of cytokines on the expression of BAFF in airwayepithelial cells. BEAS-2B cells (A) and PBEC (B) were incubated for 6 hwith 25 �g/ml dsRNA, 100 ng/ml IL-1�, 100 ng/ml IL-4, 100 ng/ml IL-6,100 ng/ml IL-10, 100 ng/ml IL-12, 100 ng/ml IL-15, 100 ng/ml TNF-�,1000 U/ml IFN-�, 100 ng/ml IFN-�, 100 ng/ml TGF-�, 100 ng/ml G-CSF,or 100 ng/ml GM-CSF, as indicated. The level of BAFF mRNA was de-termined by real-time PCR. The results are shown as the mean � SEM ofthree to four independent experiments. �, p � 0.05.

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IFN-� for up to 6 h. BAFF mRNA was strongly induced by IFN-�in a dose-dependent manner (Fig. 4A). Surprisingly, concentrationsof IFN-� as low as 1 U/ml (1 pg/ml) induced BAFF expression(Fig. 4A). Induction of mRNA for BAFF by IFN-� occurred veryrapidly, as quickly as 1 h after stimulation with IFN-� (Fig. 4B).The response to IFN-� was more rapid than the response todsRNA, which required 3 h to be apparent (Figs. 1E and 4B).These results indicate that IFN-� is a potent and rapid stimulatorof BAFF expression in airway epithelial cells.

It has been reported that IFN-� expression is also strongly in-duced by virus infection or dsRNA stimulation of airway epithelialcells (46, 47). We examined the influence of dsRNA on expressionof IFN-� in epithelial cells. Stimulation with dsRNA rapidly andstrongly induced IFN-� mRNA (Fig. 4C). If BAFF expression inresponse to dsRNA occurs as a result of induction of IFN-�, theninhibition of protein synthesis should prevent the expression ofmRNA for BAFF, but not IFN-�. BEAS-2B cells were treatedwith DMSO (vehicle control) or the protein synthesis inhibitorcycloheximide for 1 h, and then stimulated with dsRNA for 3 h.Up-regulation of mRNA for BAFF was significantly and dose de-pendently inhibited by cycloheximide (Fig. 5A). In contrast, in-duction of mRNA for IFN-� by dsRNA was not inhibited by cy-cloheximide (Fig. 5A). In fact, induction of IFN-� mRNA wasenhanced by cycloheximide, suggesting the presence of a short-lived or inducible inhibitor of the induction of IFN-�. Althoughthese results do not prove that BAFF expression is secondary to theexpression of IFN-�, they do demonstrate that BAFF is not animmediate early gene in this response, despite the fact that it israpidly induced. It has been reported that the JAK inhibitor, re-ferred to as JAK inhibitor I (JAKI), inhibits activation of STATproteins by several JAK family kinases, including JAK1, JAK2,JAK3, and tyrosine kinase 2 (Tyk2) (48, 49). Signaling induced byIFN-� via IFNAR1 and IFNAR2 is known to involve the activa-tion of STAT1 and STAT2 by JAK1 and Tyk2. To determine

whether induction of BAFF by dsRNA was mediated by an auto-crine/paracrine pathway using IFN-�, we treated BEAS-2B cellswith DMSO or JAKI for 1 h, and then stimulated with dsRNA for6 h. Induction of mRNA for BAFF by dsRNA was dose depen-dently and significantly inhibited by JAKI (Fig. 5B). Finally, toinvestigate the mechanism of BAFF induction by dsRNA morecarefully, we knocked down IFN-signaling molecules. BEAS-2Bcells were transfected with siRNA against control RNA, STAT1,or IFNAR2, and then stimulated with IFN-� and dsRNA for 6 h.Induction of BAFF by IFN-� and dsRNA was significantly inhib-ited by siRNA against STAT1 and IFNAR2, but not by controlRNA (Fig. 5C). These results suggest that BAFF is induced bydsRNA in airway epithelial cells, and that the response results viaan autocrine/paracrine pathway involving IFN-�.

Effect of glucocorticoids on induction of BAFF

Glucocorticoids are a mainstay in the treatment of diseasescharacterized by airway inflammation such as asthma, chronicobstructive pulmonary disease, and chronic rhinosinusitis (50).We investigated whether glucocorticoids were able to suppressthe dsRNA-dependent production of BAFF in airway epithelialcells. BEAS-2B cells were treated with DMSO or the potent top-ical glucocorticoid FP for 2 h, and then stimulated with dsRNA for18 h. Induction of mRNA for BAFF was significantly and dose

FIGURE 4. Effect of IFN-� on the up-regulation of BAFF by dsRNA inBEAS-2B cells. A, BEAS-2B cells were incubated for 6 h with 1–1000U/ml IFN-�. B, BEAS-2B cells were incubated with 100 U/ml IFN-� forup to 6 h. C, BEAS-2B cells were incubated with 25 �g/ml dsRNA orcontrol medium for up to 6 h. The levels of BAFF and IFN-� mRNA weredetermined by real-time PCR. The results are shown as the mean � SEMof three independent experiments.

FIGURE 5. Effect of the IFN-STAT pathway on the expression ofBAFF induced by dsRNA in BEAS-2B cells. A, BEAS-2B cells were pre-incubated with 0.01% DMSO or 0.1–10 mg/ml cycloheximide (CHX) for1 h, and then stimulated with 25 �g/ml dsRNA for 3 h. B, BEAS-2B cellswere preincubated with 0.1% DMSO or 0.1–10 �M JAKI for 1 h, and thenstimulated with 25 �g/ml dsRNA for 6 h. C, BEAS-2B cells were trans-fected with siRNA against STAT1, IFNAR2, or control RNA at 5 or 20 nMfor 72 h, and then stimulated with 1000 U/ml IFN-� and 25 �g/ml dsRNAfor 6 h. The levels of BAFF and IFN-� mRNA were determined by real-time PCR. The results are shown as the mean � SEM of four independentexperiments. �, p � 0.05 vs dsRNA or IFN-� stimulated.

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dependently inhibited by FP (Fig. 6, A and C). To confirm thisphenomenon at the protein level, we measured the production ofsBAFF (72-h culture) and mBAFF (24-h culture). We found thatinduction of both sBAFF and mBAFF by dsRNA was stronglyinhibited by FP in BEAS-2B cells (Fig. 6, D and E). In PBEC,similar results were obtained when we analyzed mRNA for BAFF.Induction of mRNA for BAFF was significantly inhibited by FP inPBEC, but to a lesser extent than in BEAS-2B cells (Fig. 6B).

Functional activity of epithelial cell-derived BAFF

It has been reported that BAFF directly promotes proliferation, cellsurvival, CSR, and Ig production in B cells through a CD40-in-

dependent and T cell-independent pathway (15, 19–21, 24). Wetherefore investigated whether epithelial cell-derived BAFF wasable to induce B cell survival using a MTT assay. We used super-natants from IFN-�-treated cells in a functional assay based uponB cell proliferation. Purified human B cells were stimulated withBAFF or concentrated supernatant in the presence or absence ofIL-4 for 5 days. Interestingly, control supernatants from epithelialcells significantly suppressed B cell proliferation. Compared withsupernatants from unstimulated epithelial cells, IFN-�-treated su-pernatants increased B cell survival (Fig. 7A). To determinewhether this effect depended on epithelial cell-derived BAFF, weused Fc fusion proteins of the BAFF-Rs, BAFF-R and TACI, toabsorb BAFF. Enhancement of cell survival by supernatants ofIFN-�-treated epithelial cells was significantly inhibited by BAFF-R:Fc and TACI:Fc, but not by control IgG1 (Fig. 7B). In contrast,anti-CD40-dependent and CpG-dependent B cell proliferation wasnot inhibited by either BAFF-R:Fc or TACI:Fc (Fig. 7B and datanot shown). These results suggest that epithelial cell-derivedBAFF is functionally active.

DiscussionThis study provides the first direct demonstration that airway ep-ithelial cells can produce the B cell-activating TNF family mem-bers, BAFF and APRIL (Figs. 1 and 2), although BAFF productionby epithelial cells from salivary glands has been published recently(45). BAFF is a centrally important regulator and activator of Bcells and has been recognized to be a main product of myeloidcells such as monocytes, macrophages, dendritic cells, neutrophils,and nurse-like cells. The quantity of BAFF produced by airwayepithelial cells in these experiments (100–200 pg/106 cells) is ofthe same order of magnitude as those produced by myeloid cells,which have been reported to generate 100–500 pg/106 cells in a72-h culture (15, 41–43).

TLRs play important roles linking innate immunity and adaptiveimmunity, and airway epithelial cells have been shown to expressfunctional TLR, notably TLR2–6 (6, 33, 34). Among the TLRligands that we tested in this study, only dsRNA, a TLR3 ligandand mimic of viral RNA and viral replication, induced BAFF ex-pression in primary bronchial epithelial cells. BAFF was also in-duced by LPS in the BEAS-2B-immortalized cell line, but not inPBEC (Fig. 1). Although epithelial cells express TLR4, and wemight have expected a more robust response to LPS, previous stud-ies have shown that production of IFN-� by LPS is regulated byserum LPS-binding protein (37). In addition, soluble CD14 is re-quired for activation of epithelial cells by LPS due to the lack ofmembrane-bound CD14 on airway epithelial cells (33, 51). In thepresent experiments, we used serum-free medium in the culture ofprimary bronchial epithelial cells, to avoid contamination with fi-broblasts. This may explain the lack of production of IFN-� andBAFF by PBEC after stimulation with LPS. The failure of otherTLR ligands tested to induce expression of BAFF may reflect lowlevels of receptor expression or adaptor proteins involved in therespective responses.

IFN-� is known to be an immediate early gene, i.e., one inwhich mRNA expression is induced without the requirement forprotein synthesis. IFN-� is directly induced by bacterial and viralPAMPs such as LPS, dsRNA, ssRNA, and CpG motif-containingDNA via TLR-dependent or TLR-independent signaling (37, 38,52–54). The IFNARs are composed of two subunits, IFNAR1 andIFNAR2, which are associated with members of the JAK family,specifically Tyk2 and JAK1, respectively (52, 55, 56). Activationby IFN-� of the JAKs that are associated with the IFNAR resultsin tyrosine phosphorylation of STAT1 and STAT2. This leads to

FIGURE 6. Effect of glucocorticoids on the up-regulation of BAFF bydsRNA in airway epithelial cells. BEAS-2B cells (A and C–E) and PBEC(B) were preincubated with 0.001% DMSO, 100 nM FP (A, B, D, and E),or 1011-106 M FP (C) for 2 h, and then stimulated with 25 �g/ml dsRNAfor 18 h (A–C), 24 h (E), or 72 h (D). The level of BAFF mRNA wasdetermined by real-time PCR (A–C). Concentrations of sBAFF protein inthe culture supernatant were measured by ELISA (D). Cells were stainedwith mAb specific for BAFF (dark line) or an isotope control mAb (shadedhistogram) and analyzed by flow cytometry (E). The results are shown asthe mean � SEM of three to four independent experiments. �, p � 0.05 vsdsRNA stimulated. **, Not detectable.

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the formation of a STAT1-STAT2-IFN regulatory factor 9 com-plex that is known as IFN-stimulated gene factor 3, and formationof STAT1-STAT1 homodimers. These complexes translocate tothe nucleus, bind to IFN-stimulated response elements (in the caseof IFN-stimulated gene factor 3) and IFN-�-activated sites (in thecase of STAT1 homodimer) in the promoter regions of relevantgenes, and initiate gene transcription (52, 55, 56). Several lines ofevidence indicate that IFN-� played a critical role in the inductionof BAFF by dsRNA in the present study: 1) dsRNA induced theexpression of IFN-� more rapidly than it induced expression ofBAFF (Figs. 1E and 4C); 2) the addition of IFN-� induced BAFFmore rapidly than dsRNA induced BAFF (Fig. 4B); 3) inhibition ofprotein synthesis by cycloheximide reduced induction of BAFF bydsRNA suggestion that it is indirect (Fig. 5A); 4) inhibition of JAKkinase by JAKI suppressed induction of BAFF by dsRNA (Fig.5B); and 5) induction of BAFF by dsRNA was significantly sup-pressed by siRNA knockdown of STAT1 and IFNAR2 (Fig. 5C).Taken together, these results suggest that dsRNA induces expres-sion of IFN-�, which in turn activates expression of BAFF. Al-though this mechanism has been demonstrated for other IFN targetgenes (52), it has not been shown for induction of BAFF bydsRNA. Litinskiy et al. (15) have shown that human dendritic cellsand monocytes up-regulate BAFF and APRIL upon stimulationwith IFN-�, IFN-�, and LPS; however, they have not described themechanism. This suggests that induction of BAFF expression bydsRNA and other PAMPs that induce IFN may be a secondaryeffect of expression of IFN-� or IFN-�.

It has been reported that sBAFF is derived from the membrane-bound form by cleavage with a putative furin family protease orderived from intracellular processing of the longer form by thesame enzyme (20, 21, 41, 42). In airway epithelial cells, produc-tion of sBAFF by dsRNA occurred in a time-dependent manner(Fig. 2). In contrast, expression of mBAFF peaked at 12–24 h afterstimulation (Fig. 2 and data not shown). Production of sBAFF bydsRNA was completely suppressed by decanoyl-Arg-Val-Lys-Arg-chloromethylketone, a specific furin convertase inhibitor (42),in BEAS-2B cells (data not shown). Furin mRNA (ID: 201945_at)was detected in BEAS-2B and primary epithelial cells in ourGeneChip analysis (data not shown). These results suggest that themechanism of production of sBAFF in airway epithelial cells maybe similar to that found in myeloid cells.

BAFF is a B cell survival factor and a costimulator of B cellproliferation and Ab secretion. Recently, Gavin et al. (39, 40) haveidentified a splice isoform of BAFF that is called �BAFF in bothmouse and human. The �BAFF transcripts of human and mouseare devoid of exons 3 and 4, respectively. �BAFF may oppose theeffects of BAFF, as �BAFF transgenic mice have been shown tohave a significant reduction in mature B cell numbers, whereasmature B cells are expanded in BAFF transgenic mice (40). The�BAFF isoform is detected in human myeloid cells, but it is aminor isoform (39). In airway epithelial cells, the �BAFF isoformwas present and induced by dsRNA, but it was only a minor iso-form (Fig. 1C). Future studies are required to determine whetherepithelial cells produce �BAFF protein.

Functional analysis showed that concentrated supernatants fromIFN-�-treated BEAS-2B cells stimulated B cell proliferationand/or survival through a mechanism that partially involves BAFF.This activity was observed despite the fact that supernatants fromunstimulated BEAS-2B cells suppressed B cell survival (Fig. 7A).The cell survival-inducing activity found in IFN-�-treated super-natants was probably due to BAFF based upon inhibition of theresponse with fusion proteins specific for BAFF-Rs (BAFF-R:Fcand TACI:Fc), but not control IgG1 (Fig. 7B). BAFF-R:Fc andTACI:Fc did not inhibit CD40-dependent proliferation and/or cellsurvival, establishing specificity (Fig. 7B). These results suggestthat the BAFF produced by dsRNA- and IFN-�-stimulated airwayepithelial cells is functionally active. The identity of the suppres-sive activity found in supernatants of unstimulated BEAS-2B isworthy of future investigation.

We examined whether glucocorticoids can inhibit dsRNA-de-pendent BAFF production, because glucocorticoids are widelyused in the therapeutic management of inflammatory airway dis-eases. In vivo studies in human subjects have demonstrated thatglucocorticoids inhibit the seasonal increase in IgE in the airwaysand in the serum of allergic individuals (57, 58). The prevailingview is that glucocorticoids achieve these effects by inhibiting theproduction of cytokines such as IL-4 and IL-13 that induce CSR,because B cell responses in vitro are resistant to glucocorticoids.As shown in Fig. 6, the potent topical glucocorticoid FP signifi-cantly inhibited induction of both BAFF mRNA and BAFF proteinby dsRNA in BEAS-2B cells. Expression of BAFF mRNA in-duced by dsRNA in PBEC was also inhibited by FP (Fig. 6B).

FIGURE 7. Effect of epithelial cell-derivedBAFF on the cell survival of B cells. A, Purifiedhuman B cells were stimulated with 5 ng/mlBAFF or concentrated BEAS-2B supernatant for5 days. B, Purified human B cells were stimu-lated with 5 ng/ml BAFF, 10 ng/ml IL-4, 10�g/ml mouse anti-human CD40 mAb, concen-trated supernatant, or the indicated combinationsfor 5 days in the presence or absence of 5 �g/mlcontrol IgG1, 5 �g/ml BAFF-R:Fc, or 5 �g/mlTACI:Fc. MTT reagent was added for the last4 h of incubation. The concentrated BEAS-2Bsupernatant was added at a final dilution of 1/20to the B cell culture. Absorbance at 570 nm wasmeasured using a microtiter plate reader. The re-sults are shown as the mean � SEM of five in-dependent experiments. �, p � 0.05.

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Future clinical studies are required to investigate whether glu-cocorticoids suppress BAFF induction in vivo.

Local activation of B cell proliferation, CSR, and Ab productionis likely to be of great importance in immunity of the airways topathogens. In asthma, disease exacerbations are usually triggeredby infection with airway-targeting viruses, especially rhinovirus,respiratory syncytial virus, influenza virus, and adenovirus (59–61). The ability of the local tissue to mount a rapid and effectivelocal Ig response against virus is most likely to be important in thelimitation of the extent and severity of virus-induced tissue dam-age. Local expression of germline transcripts and excision circlesfor IgE and activation-induced cytidine deaminase, essential reg-ulators of CSR, has been demonstrated to occur in the airwaymucosa following nasal challenge with allergen or diesel exhaustextracts (9–11, 62, 63). B cell clusters are found in the laminapropria of the nasal mucosa (11, 64). IgE-expressing B cells andplasma cells are found in the lamina propria, but not in the epi-thelium (64). IgA and IgE are also found in the bronchial lavageand in the nasal lavage of allergic patients (65, 66). The levels ofAg-specific IgE are frequently found to be much higher in theairways and in the nasal mucosa than they are in the serum (65–67). Thus, the local CSR and activation of B cells may be an eventthat is important both in disease pathogenesis and protection frompathogens. Our data show that BAFF protein is detected on boththe apical and basolateral sides of cultured epithelial cells. Ourresults suggest that local activation of B cells in the airways maydepend in part on epithelial production of BAFF and APRIL. Ininflammatory lung diseases, activated airway epithelial cells pro-duce chemokines that recruit many cell types capable of producingtype I IFN, including monocytes, macrophages, and dendritic cells(50, 68). These recruited cells produce IFNs directly and also pro-duce BAFF via autocrine pathways involving IFNs when stimu-lated with PAMPs and cytokines (15). Production of IFN by in-filtrating inflammatory cells could activate epithelial cells toproduce BAFF in vivo.

Recently, Sutherland et al. (69) have shown that BAFF trans-genic mice have suppressed OVA-induced allergic airway re-sponses, including reduced eosinophils in BALF and infiltratingleukocytes around airways and pulmonary blood vessels. The stud-ies of Sutherland et al. (69) indicate that high levels of systemicBAFF alter T cell homing to the airways, not T cell priming or Bcell responses. Studies targeting BAFF production in the airwayswill be necessary to determine whether local BAFF produced inthe lungs or upper airways can contribute meaningfully to local Bcell responses. In a separate clinical study, we have detectedmRNA for BAFF and APRIL in nasal scraping epithelial cells andfound increased expression of BAFF protein in nasal tissue ex-tracts and nasal lavage from patients with chronic rhinosinusitis(A. Kato, A. T. Peters, R. Kern, D. Conley, L. C. Grammer, andR. P. Schleimer, manuscript in preparation).

In summary, we report in this study that airway epithelial cellsproduce the B cell-activating factors, BAFF and APRIL. Our find-ings indicate that BAFF is induced by cytokines and dsRNA inairway epithelial cells, and that the response to the latter resultsfrom an IFNAR-mediated autocrine/paracrine pathway of IFN-�.The production of BAFF and APRIL by epithelial cells may con-tribute to local accumulation, activation, CSR, and Ig synthesis byB cells in the airway.

AcknowledgmentsWe thank Drs. Marianna Kulka and Ning Zhang and Brian Tancowny ofNorthwestern University Feinberg School of Medicine for technical advicewith flow cytometry, real-time PCR, and PBEC preparation. We thankAnne Jedlicka and the Malaria Research Institute Gene Array Core Facility

at the Johns Hopkins University Bloomberg School of Public Health fortheir roles in the gene array experiments.

DisclosuresDr. Schleimer has received honoraria from all major manufacturers of in-haled glucocorticoids, including GlaxoSmithKline, the manufacturer offluticasone propionate.

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