Epithelial cells trigger frontline immunoglobulin class ... - Nature

© 2007 Nature Publishing Group http://www.nature.com/natureimmunology

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Epithelial cells trigger frontline immunoglobulin class switching through a pathway regulated by the inhibitor SLPI Weifeng Xu1, Bing He1, April Chiu1, Amy Chadburn1, Meimei Shan2, Malwina Buldys1, Aihao Ding2,3, Daniel M Knowles1, Paul A Santini1 & Andrea Cerutti1,3 Epithelial cells (ECs) transport class-switched immunoglobulin G (IgG) and IgA antibodies across mucous membranes. Whether ECs initiate class switching remains unknown. Here we found that ECs lining tonsillar crypts formed pockets populated by B cells expressing activation-induced cytidine deaminase (AID), an enzyme associated with ongoing class switching. ECs released B cell–activating AID-inducing factors after sensing microbial products through Toll-like receptors. The resulting class switching was amplified by thymic stromal lymphopoietin, an epithelial interleukin 7–like cytokine that enhanced the B cell ‘licensing’ function of dendritic cells, and was restrained by secretory leukocyte protease inhibitor, an epithelial homeostatic protein that inhibited AID induction in B cells. Thus, ECs may function as mucosal ‘guardians’ orchestrating frontline IgG and IgA class switching through a Toll-like receptor–inducible signaling program regulated by secretory leukocyte protease inhibitor.

Epithelial cells (ECs) lining the respiratory, gastrointestinal and urogenital mucous membranes separate the sterile internal milieu of the body from the contaminated external environment. By blocking the passive movement of commensal bacteria, invasive pathogens, toxins and other noxious agents into the subepithelial environment, ECs prevent the onset of local and systemic inflammation and provide a first line of defense against infection1–4. In addition to serving as a physical barrier, ECs participate in mucosal immune responses by sensing the presence of commensal and pathogenic microorganisms through a vast array of pattern-recognition receptors, including Tolllike receptors (TLRs)5,6. Engagement of TLRs on ECs by microbial molecular patterns generates homeostatic signals essential for the epithelial barrier to maintain its physical integrity7. Signals emanating from TLRs also stimulate ECs to release small proteins with antimicrobial function, including defensins, cathelicidins, angiogenins, secretory leukocyte protease inhibitor (SLPI) and elafin6,8–10. Those innate effector molecules help ECs to control the growth of commensal and pathogenic bacteria and to promote the recruitment and maturation of antigen-presenting dendritic cells (DCs)11,12. ECs further intersect the innate immune system by providing chemoattractant signals to neutrophils and other inflammatory cells at mucosal sites of infection. ECs modulate mucosal adaptive immune responses by participating in an intimate crosstalk with DCs13. Those DCs sample lumenal antigens and present them to subepithelial CD4+ T helper cells to initiate adaptive T helper type 2 (TH2) responses characterized by the

production of interleukin 4 (IL-4) and IL-10 (refs. 14,15). In addition to regulating mucosal immune homeostasis through their antiinflammatory properties, TH2 cytokines stimulate mucosal B cells to produce protective antibodies16. Mucosal DCs acquire TH2stimulating functions by interacting with thymic stroma lymphopoietin (TSLP), an IL-7-like cytokine produced by ECs15,17,18, suggesting that a crosstalk between ECs and DCs modulates both the cellular and humoral arms of the mucosal immune system. Mucosal TH2 cells activate antigen-specific B cells through CD40 ligand (CD40L) and cytokines such as IL-4 and IL-10 (ref. 19). Activated B cells undergo immunoglobulin class-switch recombination (CSR) and somatic hypermutation in the germinal centers of mucosal lymphoid follicles20. CSR substitutes immunoglobulin M (IgM) and IgD with IgG, IgA or IgE, thereby endowing antibodies with distinct effector functions that enhance antigen clearance21. Somatic hypermutation introduces point mutations in the variable genes encoding the antigen-binding region of immunoglobulins, thereby providing the structural correlate for selection by antigen of mutants with higher affinity22. Ultimately, germinal center B cells differentiate into plasma cells23. Those effector B cells are recruited by ECs to the subepithelial area, where they secrete monoreactive antibodies to microbes19,24,25. ECs can also recruit ‘preswitched’ B cells26. Such cells can rapidly undergo CSR and antibody production in the subepithelial area27, possibly after interacting with antigen-loaded DCs expressing B cell– activating factor (BAFF; also called BLyS) of the tumor necrosis factor family and IL-10 (refs. 16,28–31). Low-affinity and broadly reactive

1Department of Pathology and Laboratory Medicine, 2Department of Immunology and Microbiology and 3Graduate Program of Immunology and Microbial Pathogenesis, Weill Medical College of Cornell University, New York, New York 10021, USA. Correspondence should be addressed to A.C. ([email protected]).

Received 12 September 2006; accepted 21 December 2006; published online 28 January 2007; corrected online 8 February 2007 (details online); doi:10.1038/ni1434

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IgM, IgG and IgA antibodies emerging from that CD40-independent pathway would be involved in the steady-state control of commensal bacteria and provide a first line of defense against pathogens16,32,33. Ultimately, IgM, IgG and IgA generated through either CD40dependent or CD40-independent pathways are translocated onto the mucosal surfaces by ECs by transcytosis34–39. Although B cell recruitment and transcytosis are well established contributions of ECs to mucosal humoral immunity, it is unknown whether ECs can initiate class switching and antibody production. Here we report that ECs lining tonsillar crypts formed pockets colonized by B cells expressing activation-induced cytidine deaminase (AID), an enzyme associated with ongoing CSR16,40. In the presence of TLR-binding microbial products, ECs released BAFF and IL-10 and thereafter stimulated B cells to express AID, undergo CSR and ultimately secrete polyreactive IgG and IgA antibodies to multiple microbial determinants. EC-induced class switching was enhanced by TSLP, an epithelial cytokine that elicited BAFF production by DCs, but was restrained by SLPI, a homeostatic epithelial protein that inhibited BAFF signaling in B cells. Our data indicate that intraepithelial pockets in the aerodigestive mucosa promote frontline IgG and IgA class switching through a TLR-dependent SLPI-regulated signaling program linking ECs with DCs and B cells. RESULTS Intraepithelial B cells express AID Human tonsils are secondary lymphoid tissues that mount protective IgG and IgA responses to commensal and pathogenic microorganisms present in the aerodigestive tract4. Unlike the lower respiratory and digestive tracts, which are lined by a single layer of epithelial cells, the tonsillar mucosa is lined by a stratified epithelium that comprises two distinct compartments. The surface epithelium has a squamous morphology and lines the external portion of the tonsil to protect it from antigen entry4,41. The crypt epithelium has a reticular morphol-

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Figure 1 Tonsillar ECs express the AID-inducing ligand BAFF and form mucosal pockets infiltrated by AID-expressing B cells. (a) Immunofluorescence analysis of reticular epithelium from a tonsillar crypt stained for CD11c (green), Pax5 (red) and cytokeratin (blue). (b) Reticular epithelium stained for CD123 (green), Pax5 (red) and cytokeratin (blue). Right, detail of intraepithelial B cells at left. (c) Reticular epithelium stained for cytokeratin (green), AID (red) and IgD (blue). (d) Interfollicular epithelium and subepithelial secondary lymphoid follicles stained for cytokeratin (green), AID (red) and IgD (blue). Arrowheads indicate AID+ B cells in interfollicular epithelium. (e) Reticular epithelium and subepithelial area stained for IgD (green), cytokeratin (red) and BAFF (blue). (f) Reticular epithelium stained for IgD (green), cytokeratin (red) and BAFF (blue). Arrowheads indicate intraepithelial IgD+ B cells proximal to the lumen of the crypt. (g) Squamous epithelium and subepithelial area stained for IgD (green), cytokeratin (red) and BAFF (blue). Original magnification, 5 (a, and b, left) 40 (b, right), 60 (c), 10 (d), 20 (e,g) or 40 (f). Data are from one of ten tissue samples yielding similar results.

ogy and lines tonsillar invaginations serving as active sites of antigen sampling and entry4,41. Because of its continuous exposure to viruses and bacteria, the crypt epithelium is extensively infiltrated by lymphocytes, including B cells4,41. As class-switched IgG and IgA antibodies are key to mucosal immunity4,20,33, we hypothesized that tonsillar crypts provide B cells with an epithelial niche to initiate frontline class switching. We found that cytokeratin-positive ECs in the reticular epithelium of tonsillar crypts formed pockets filled with B cells expressing Pax5 (also called B cell–specific activation protein), a B cell–restricted nuclear protein that regulates immunoglobulin genes21 (Fig. 1a,b and Supplementary Fig. 1 online). Consistent with their key involvement in mucosal immunity13,42,43, CD11c+ myeloid DCs and CD123+ plasmacytoid DCs colonized epithelial pockets together with B cells. As DCs can induce CSR29,44, we reasoned that some intraepithelial B cells could express AID, an enzyme associated with ongoing CSR40. Consistent with that possibility, scattered B cells in the reticular epithelium expressed AID but not IgD (Fig. 1c), a phenotype similar to that of B cells actively undergoing CSR in the germinal center (Fig. 1d). Intraepithelial IgD–AID+ B cells were proximal to subepithelial IgD+AID– B cells, suggesting that intraepithelial B cells undergoing CSR originate in situ from a preswitched precursor. ECs produce the class switch–inducing factor BAFF DCs upregulate AID expression and trigger CSR by stimulating B cells through BAFF16,29,30,44. Having found extensive infiltration of the crypt epithelium by both myeloid and plasmacytoid DCs, we hypothesized that abundant intraepithelial BAFF would be present. The reticular epithelium lining tonsillar crypts contained abundant BAFF. Unexpectedly, most of that BAFF was in cytokeratin-positive ECs (Fig. 1e), although some BAFF was also present in cytokeratinnegative DCs (Fig. 1f). Cytokeratin-postitive BAFF+ ECs enveloped IgD+ B cells colonizing the crypt epithelium and were proximal to IgD+ B cells lodged under the surface epithelium (Fig. 1g). These data suggested that aerodigestive ECs have the potential to initiate frontline class switching through an innate pathway comprising BAFF. ECs release BAFF in response to viral RNA ECs release immune modulators after sensing microbial products through TLRs6. Thus, we sought to determine if ECs produce BAFF in response to TLR ligands. Functional studies involving tonsillar ECs are limited by poor yield, heavy microbial contamination, low viability, the presence of DCs and reduced responsiveness to in vitro stimuli as a result of continuous in vivo activation. A suitable alternative is provided by ECs from the oral cavity, which constitute

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an integral component of the aerodigestive c 30 a 250 b 24 mucosa together with tonsillar ECs. The oral 25 20 200 epithelium forms a continuum with and is 20 16 150 15 12 morphologically and phenotypically identical 100 10 8 to the tonsillar epithelium. Preliminary data 50 5 4 showed that serially propagated ECs from the 0 0 0 oral mucosa lacked contaminating DCs (Supplementary Fig. 1) and were thus suitable for the study of EC-induced B cell responses. TLR3 f d e lgD Consistent with that possibility, oral ECs not only contained BAFF but further upre5 lgD Cytokeratin TLR3 gulated this innate class switch-inducing 4 factor after exposure to polyinosinic3 2 polycytidylic acid (poly(I:C); Supplementary 1 Fig. 1), a synthetic analog of viral RNA that lgD TLR3 DAPI 0 binds TLR3 (ref. 5). Therefore, we used oral ECs as an inducible model for studying the class switch–inducing function of the aeroB B + ECs digestive epithelium. In the presence of poly(I:C), ECs upregug5 i 75 lated the expression of TNFSF13B mRNA, * h 5 * * which encodes BAFF, as well as the release of 4 4 60 soluble BAFF and IL-10 proteins (Fig. 2a–c). 3 3 45 * * * 2 2 30 We also found induced upregulation of * * * * * 1 1 15 * TNFSF13B, BAFF and IL-10, although less 0 0 0 so than with poly(I:C), by peptidoglycan Iγ3-Cµ Iµ-Cµ (PGN), a bacterial wall component that binds TLR1, TLR2 and TLR6 (ref. 5); B B + ECs B B + ECs lipopolysaccharide (LPS), a bacterial wall B B + ECs component that binds TLR4 (ref. 5); hypomethylated deoxycytidylate-phosphateFigure 2 ECs release BAFF and IL-10 and induce AID expression as well as IgG and IgA class deoxyguanylate oligodeoxynucleotide (CpG switching in B cells after sensing viral RNA. (a) Real-time RT-PCR of TNFSF13B transcripts from oral ODN), a viral DNA analog that binds TLR9 ECs cultured for 24 h with medium alone, LPS, PGN, CpG ODN, poly(I:C) or IFN-a. TNFSF13B mRNA (ref. 5); and interferon-a (IFN-a), a BAFF- is normalized to ACTB mRNA. (b,c) ELISA of BAFF (b) and IL-10 (c) from oral ECs cultured for 48 h inducing cytokine secreted by plasmacytoid as described in a. (d) Immunofluorescence analysis of surface epithelium and crypt epithelium from DCs29,43. The general relevance of TLR3 in a tonsil stained for IgD (green), cytokeratin (red) and TLR3 (blue). (e) Immunofluorescence analysis EC-mediated BAFF-dependent class switch- of subepithelial B cells stained for IgD (green), TLR3 (red) and DAPI (blue). Original magnification, 5 (d) or 40 (e). (f–h) Quantitative real-time RT-PCR of TLR3 (f), AICDA (g) and Ig3-Cm (h) transcripts ing was suggested by the detection of from peripheral blood IgD+ B cells incubated for 48 h with or without IL-10 and/or poly(I:C) in the abundant TLR3 not only in the stratified presence (filled bars) or absence (open bars) of oral ECs. TLR3 and AICDA mRNA is normalized to epithelium of tonsils (Fig. 2d) but also in ACTB mRNA; Ig3-Cm mRNA is normalized to Im-Cm mRNA, which is constitutively expressed by B cells. the stratified epithelium of the epidermis Below h, gel electrophoresis of RT-PCR-amplified Ig3-Cm transcripts and Im-Cm transcripts (loading and in the nonstratified epithelium of the control). (i) ELISA of IgG antibodies secreted by peripheral blood IgD+ B cells cultured for 8 d as lower digestive tract (Supplementary Fig. 2 described in f–h. Data are one of three experiments yielding similar results (a–f) or summarize three online), as well as by the observation that, experiments (g–i; error bars, s.d.; *, P o 0.05). like oral ECs, epidermal and intestinal ECs augmented the expression of TLR3 and TNFSF13B transcripts as well DCs and B cells. Given that TLR3 was abundantly expressed by ECs as the production of soluble BAFF protein in response to poly(I:C) and that poly(I:C) was the most effective inducer of BAFF production (Supplementary Fig. 2). In addition to TLR3, tonsillar, epidermal and by ECs, we hypothesized that TLR3 engagement by viral RNA initiates intestinal ECs expressed other BAFF-inducing TLRs, including TLR9 frontline class switching by linking ECs to B cells through BAFF. (Supplementary Fig. 3 online). Those ECs and oral ECs augmented Consistent with that hypothesis, we detected TLR3 not only in the expression of TLR9 and TNFSF13B transcripts as well as the cytokeratin-positive ECs but also in subepithelial IgD+ B cells production of soluble BAFF protein in response to CpG ODN (Fig. 2e) and interfollicular CD123+BAFF+ DCs from the tonsillar (Supplementary Fig. 3 and data not shown). These findings indicated mucosa (Supplementary Fig. 4 online). As peripheral blood B cells that ECs from multiple frontline areas can release innate class switch– had variable but overall low expression of TLR3 (Supplementary inducing molecules, such as BAFF, after sensing microbial products Fig. 4), we hypothesized that B cells colonizing the tonsillar mucosa through TLRs. upregulate TLR3 as a result of their exposure to local stimuli. Accordingly, resting peripheral blood IgD+ B cells upregulated their BAFF increases B cell responsiveness to viral RNA expression of TLR3 after exposure to poly(I:C) alone or combined Having found that B cells and DCs populated both the epithelial and with EC- or DC-derived factors, including BAFF, IL-10 and IFN-a subepithelial areas of the aerodigestive tract, we reasoned that local (Fig. 2f and Supplementary Fig. 4). Poly(I:C) upregulated TLR3 also antibody responses could involve intimate interaction of ECs with in the presence of engagement of the B cell antigen receptor (BCR).

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Figure 3 ECs exposed to viral RNA induce class switching by activating B cells through BAFF. (a) RT-PCR of TNFSF13B and ACTB and immunoblot analysis of BAFF and actin from oral ECs exposed to no siRNA, control siRNA or siRNA targeting BAFF. (b) Quantitative RT-PCR of AICDA and ELISA of IgG, IgA and IgM antibodies from IgD+ B cells incubated with ECs not treated with siRNA (ECs), BAFF-sufficient oral ECs exposed to irrelevant siRNA (Control ECs) or BAFF-deficient oral ECs exposed to siRNA targeting BAFF (BAFF KD ECs) in the presence (+) or absence (–) of poly(I:C) and IL-10. Cells were cultured for 48 h for AICDA analysis and for 8 d for immunoglobulin analysis. AICDA mRNA is normalized to ACTB mRNA. (c) AICDA transcripts and IgG and IgA proteins from IgD+ B cells exposed to oral ECs in the presence or absence of poly(I:C) and IL-10 plus either control immunoglobulin or BCMA-Ig. (d) In situ RNA hybridization of AICDA transcripts in the reticular epithelium from a tonsillar crypt. DAPI stains nuclei of ECs. Dashed lines outline two representative intraepithelial pockets with AICDA-expressing B cells. (e) Immunofluorescence analysis of crypt epithelium and a subepithelial secondary lymphoid follicle from a tonsil stained for IgG (green) and IgD (blue). Original magnification, 40 (d) or 5 (e). Data are one of three experiments yielding similar results (a,d,e) or summarize three experiments (b,c; error bars, s.d.; *, P o 0.05).

TLR3 was functional in tonsillar B cells, as these cells initiated activation of the CSR-inducing factor NF-kB21 in the presence of poly(I:C) (Supplementary Fig. 4). Thus, viral RNA triggers complex crosstalk among ECs, DCs and B cells. Such crosstalk would involve BAFF and may render B cells more responsive to viral RNA through a mechanism involving upregulation of TLR3. ECs stimulate class switching after sensing viral RNA We further elucidated the functional cooperation between ECs and B cells in a simplified three-dimensional in vitro mucosa model comprising IgD+ B cells and ECs with a spatial distribution similar to that found in vivo. We seeded ECs in the upper chamber of a transwell filter and placed B cells in the lower chamber. To mimic viral infection, we added poly(I:C) to ECs in the upper chamber. To compensate for the lack of subepithelial DCs producing IL-10, we supplemented B cells in the lower chamber with IL-10. IgD+ B cells incubated with poly(I:C) and IL-10 in the presence of ECs had more expression of AICDA transcripts (encoding AID) and of switch Ig-Cm and Ia-Cm circular transcripts (molecular byproducts of IgG and IgA CSR, respectively29,40) than did IgD+ B cells incubated with poly(I:C) and IL-10 in the absence of ECs (Fig. 2g,h and Supplementary Fig. 5 online) In addition, ECs enhanced the poly(I:C)- and IL-10-induced production of IgG and IgA antibodies (Fig. 2i and Supplementary Fig. 5). Those antibodies increased further in the presence of BCR engagement (data not shown). Epidermal ECs, which are morphologically and phenotypically similar to tonsillar and oral ECs, induced similar IgG and IgA responses, including the production of broadly reactive IgG antibodies to influenza virus, bacterial LPS, bacterial phosphorylcholine and double-stranded DNA (Supplementary Fig. 6). Of note, ECs did not augment the production of polyreactive

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IgM antibodies over the amounts already induced by poly(I:C) and IL-10 (Supplementary Fig. 6). These data indicated that frontline ECs induce IgG and IgA CSR and production after sensing viral RNA. ECs induce class switching by stimulating B cells through BAFF BAFF is an innate class switch–inducing factor usually produced by DCs29. To ascertain whether BAFF is linked to EC-induced class switching, we resorted to ‘knocking down’ BAFF expression in ECs through RNA interference. ECs transfected with small interfering RNA (siRNA) targeting TNFSF13B had less TNFSF13B transcript and less BAFF protein expression than did ECs exposed to no siRNA or to control siRNA, but had similar expression of ACTB transcripts (encoding b-actin) and actin protein (Fig. 3a). In the presence of poly(I:C), BAFF-deficient ECs induced less AICDA expression and less IgG and IgA production in IgD+ B cells than did BAFF-sufficient ECs (Fig. 3b). Of note, lack of BAFF did not prevent ECs from inducing IgM secretion. BAFF-sufficient and BAFF-deficient epidermal or intestinal ECs yielded results similar to those obtained with oral ECs data not shown). The key involvement of BAFF in EC-induced class switching was further supported by experiments showing that the soluble BAFF ‘decoy receptor’ BCMA-Ig (B cell maturation antigen– immunoglobulin) inhibited the EC-induced production of AICDA transcripts as well as the secretion of IgG and IgA proteins by IgD+ B cells, whereas a control immunoglobulin did not (Fig. 3c). The in vivo relevance of those in vitro data was confirmed by the detection of AICDA transcripts (Fig. 3d) as well as IgG proteins (Fig. 3e) and IgA proteins (data not shown) in B cells lodged in intraepithelial pockets of tonsillar crypts. These findings indicated that ECs induce the CSR machinery, including AID, and subsequently trigger IgG and IgA class switching by activating B cells through BAFF.

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Figure 4 Tonsillar ECs interact closely with BAFF-expressing DCs and upregulate the DC-stimulating cytokine TSLP in response to viral RNA. (a) Immunofluorescence analysis of reticular epithelium from a tonsillar crypt stained for IgD (green), BAFF (red) and TSLP (blue). (b) Immunofluorescence analysis of the subepithelial area of a tonsil stained for IgD (green), TLR3 (red) and BAFF (blue). (c) Immunofluorescence analysis of subepithelial DCs stained for CD123 or CD11c (red) and BAFF (blue). Original magnification, 5 (a) or 40 (b,c). (d) Real-time RT-PCR of TSLP mRNA and ELISA of TSLP protein from ECs incubated for 24 h (RT-PCR) or 48 h (ELISA) with medium alone, LPS, PGN, CpG ODN, poly(I:C) or IFN-a. TSLP mRNA is normalized to ACTB mRNA. Data are one of three experiments yielding similar results.

ECs stimulate BAFF production by DCs through TSLP Having found that ECs closely interacted with B cells and DCs and knowing that DCs trigger class switching through BAFF29 and receive activating signals from ECs through TSLP45, we sought to determine whether ECs amplify intraepithelial class switching by stimulating DCs to produce more BAFF via TSLP. In addition to BAFF+CD123+ plasmacytoid DCs, the subepithelial area of the tonsillar mucosa included BAFF+CD11c+ myeloid DCs, which closely interacted with TSLP+ ECs and with IgD+ B cells (Fig. 4a–c). ECs upregulated the expression of TSLP transcripts and released soluble TSLP protein after exposure to poly(I:C) (Fig. 4d). In contrast, LPS, PGN, CpG ODN and IFN-a only marginally increased TSLP production. Epidermal and intestinal ECs yielded results similar to those obtained with oral ECs (data not shown). In the presence of TSLP, monocyte-derived myeloid

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DCs upregulated constitutive and/or poly(I:C)-induced expression of TLR3 transcripts, CRLF2 transcripts (encoding cytokine receptor–like factor 2, also known as the TSLP receptor) and TNFSF13B transcripts (Fig. 5a–c and Supplementary Fig. 7 online), suggesting the existence of crosstalk among viral RNA, TSLP and BAFF. In addition, myeloid DCs upregulated constitutive as well as poly(I:C)-induced expression of membrane-bound BAFF, soluble BAFF and IL-10 after exposure to TSLP (Fig. 5d–f), further indicating that intraepithelial class switching stems from intimate interaction of ECs with DCs and B cells.

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Figure 5 TSLP amplifies EC-induced class switching by stimulating DCs to produce more BAFF. (a–c) Real-time RT-PCR of TLR3 mRNA (a), CRLF2 mRNA (b) and TNFSF13B mRNA (c) from DCs incubated for 24 h with or without poly(I:C) in the presence (open bars) or absence (filled bars) of TSLP. TLR3, CRLF2 and TNFSF13B mRNA is normalized to ACTB mRNA. (d) Flow cytometry of membrane-bound BAFF (mBAFF) on DCs cultured as described in a–c. (e,f) ELISA of soluble BAFF (sBAFF) and IL-10 from DCs cultured as described in a–c. (g) ELISA of total IgG antibodies from peripheral blood IgD+ B cells incubated for 8 d with or without poly(I:C) and in the presence or absence of ECs alone, DCs alone or a combination of ECs and DCs. DC–B cell cocultures are with or without TSLP, control immunoglobulin and/or BCMA-Ig; EC–DC–B cell cocultures are with or without control Ig, blocking antibody to TSLP and/or BCMA-Ig. Data summarize three experiments (error bars, s.d.; *, P o 0.05, and **, P o 0.005).

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IgD DAPI SLPI Merged Figure 6 Tonsillar ECs express SLPI and form intraepithelial pockets containing SLPI+ B cells. (a) Immunofluorescence analysis of 20 16 reticular epithelium from a tonsillar crypt stained for SLPI (green), 12 Pax5 (red) and TSLP (blue). (b) Crypt epithelium stained for 8 cytokeratin (green), SLPI (red) and immunoglobulin (blue). 4 0 Arrowheads point to SLPI+ intraepithelial B cells. (c) Reticular Time (h) 0 1 2 3 4 6 8 16 24 48 epithelium stained for AID (green), BAFF (red) and SLPI (blue). Poly(I:C) (d) Real-time RT-PCR of SLPI mRNA from ECs incubated for 24 h with medium alone, LPS, PGN or CpG ODN in the presence or absence of poly(I:C). SLPI mRNA is normalized to ACTB mRNA. (e) Real-time RT-PCR of SLPI, TNFSF13B and TSLP transcripts from ECs incubated with poly(I:C) (time, horizontal axis). SLPI, TNFSF13B and TSLP mRNA is normalized to ACTB mRNA. (f) Immunofluorescence analysis of resting peripheral blood IgD+ B cells exposed for 2 h to medium alone or SLPI, washed extensively and subsequently stained for IgD (green), SLPI (red) and DAPI (blue). Original magnification, 5 (a) or 40 (b,c,f). Data are one of three experiments yielding similar results. SLPI


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and myeloid DCs in the lower chamber. In the presence of poly(I:C), myeloid DCs induced as much IgG production as did ECs (Fig. 5g). When combined with TSLP, myeloid DCs further increased their B cell–stimulating activity and induced as much IgG production as a combination of myeloid DCs and ECs. Soluble BCMA-Ig decoy receptor inhibited the induction of IgG production by myeloid DCs or by a combination of ECs and myeloid DCs, whereas a control immunoglobulin with irrelevant binding activity did not. Similarly, a blocking antibody to TSLP attenuated the induction of IgG production by ECs and myeloid DCs, whereas a control antibody with irrelevant binding activity did not. Epidermal and intestinal ECs yielded results similar to those obtained with oral ECs (data not shown). These findings indicated that ECs amplify the class switch– inducing activity of myeloid DCs by augmenting their ability to produce BAFF through TSLP. ECs upregulate SLPI expression in response to viral RNA ECs produce homeostatic proteins that control mucosal inflammation10. Of those proteins, SLPI is a small ‘alarm’ antiprotease that prevents LPS from activating macrophages through NF-kB46–48. Given that BAFF needs NF-kB to induce CSR29, we reasoned that SLPI could be important for ECs to restrain their BAFF-dependent class switchinducing activity. We detected SLPI in the cytoplasm and, to a larger extent, in the nuclei of cytokeratin-positive TSLP+BAFF+ ECs from both crypt and surface epithelia of tonsils (Fig. 6a–c and Supplementary Fig. 8 online). We mixed SLPI+ ECs together with Pax5+ and Ig+ B cells, including AID+ B cells. Like ECs, some intraepithelial B cells contained SLPI in both cytoplasm and nucleus. Poly(I:C) induced expression of SLPI transcripts in ECs more effectively than did LPS or PGN (Fig. 6d). In addition, poly(I:C) enhanced the SLPI-inducing activity of CpG ODN. Consistent with its possible regulatory function in EC-induced class switching, SLPI transcripts peaked at 48 h after exposure of ECs to poly(I:C), whereas TNFSF13B and TSLP transcripts peaked after 6 h and 3 h, respectively (Fig. 6e). Thus, ECs produce a homeostatic regulator of mucosal class switching after sensing viral RNA. SLPI attenuates class switching in BAFF-induced B cells SLPI released by ECs can penetrate macrophages48. Thus, we sought to determine whether SLPI also penetrates B cells. IgD+ B cells incubated

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with SLPI stained positive for SLPI in both the cytoplasm and nucleus (Fig. 6f). Given its ability to inhibit NF-kB48, a key CSR-inducing transcription factor21, SLPI might attenuate CSR in B cells. CSR from Cm to a downstream Cg3, Cg1, Ca1, Cg2, Cg4, Ca2 and Ce gene is guided by switch (S) regions 5¢ of each heavy-chain constant gene (CH) and 3¢ of a heavy-chain intronic exon (IH)22. CSR is preceded by germline IH-S-CH transcription, a key event that requires activation of the IH promoter flanking each IH exon21. In addition to yielding ‘sterile’ IH-CH transcripts, germline IH-S-CH transcription enables recruitment of the CSR machinery, including AID, to the targeted S region49. SLPI impaired poly(I:C)-, BAFF- and IL-10-induced transcriptional activation of the Ig3 promoter in IgD+ 2E2 B cells (Fig. 7a), a subclone of the human CL-01 B cell line50. In similarly stimulated IgD+ B cells, SLPI inhibited the induction of germline Ig3-Cg3 and Ig1-Cg1 transcripts and the induction of AICDA transcripts as well as the production of IgG and IgA antibodies (Fig. 7b–d). Of note, SLPI attenuated also IgG and IgA production induced by CD40L and IL-10 (Supplementary Fig. 9 online). Those inhibitory effects were specific, as SLPI had no inhibitory effect on IgM production and did not inhibit the survival and proliferation of B cells (Fig. 7e–i and Supplementary Fig. 9). Thus, SLPI dampens IgG and IgA CSR in B cells exposed to viral RNA and to EC-derived products such as BAFF and IL-10. SLPI attenuates NF-jB activation in BAFF-induced B cells BAFF and TLR ligands initiate CSR by activating IH promoters through NF-kB proteins, including p50, p65 and c-Rel29,50. Thus, we verified whether SLPI interferes with the activation of NF-kB in B cells exposed to viral RNA and EC-derived products, including BAFF. SLPI inhibited the transcriptional activation of an NF-kB-dependent minimal promoter in IgD+ 2E2 B cells exposed to poly(I:C), BAFF and IL10 (Fig. 7j). In addition, SLPI inhibited the nuclear translocation of p50, p65 and c-Rel and the binding of nuclear p50, p65 and c-Rel to DNA in IgD+ B cells exposed to poly(I:C), BAFF and IL-10 (Fig. 7k,l). In those cells, SLPI did not affect the expression of nuclear Oct-1, a ubiquitous nuclear transcription factor that regulates immunoglobulin gene expression21, and the binding of nuclear Oct-1 to DNA. These findings indicated that SLPI inhibits CSR in B cells exposed to viral RNA and EC-derived products, such as BAFF and IL-10, by interfering with the activation of NF-kB. Thus, we propose a model in which microbial products trigger intraepithelial class switching by linking ECs

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8 6 4 2

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Iγ3-Luc (‘fold induction’)

a

Figure 7 SLPI negatively regulates class switching in B cells exposed to viral RNA, BAFF and IL-10. (a) Luciferase activity of IgD+ 2E2 B cells transfected with Ig3-Luc and cultured for 48 h with or without poly(I:C), BAFF and IL-10 in the presence (filled bars) or absence (open bars) of SLPI. (b–d) Real-time RT-PCR of Ig1-Cg1 mRNA (b), Ig3-Cg3 mRNA (c) or AICDA mRNA (d) from peripheral blood IgD+ B cells cultured for 4 d as described in a. Ig1-Cg1, Ig3-Cg3 and AICDA mRNA is normalized to ACTB mRNA. (e–g) ELISA of total IgM, IgG and IgA antibodies secreted by IgD+ B cells cultured for 8 d as described in a. (h) Proportion of peripheral blood IgD+ B cells binding the apoptotic marker annexin V after 4 d of culture as described in a. (i) [3H]thymidine incorporation by peripheral blood IgD+ B cells cultured for 4 d as described in a. (j) Luciferase activity of IgD+ 2E2 B cells transfected with kB(2)-Luc and cultured for 48 h as described in a. (k) Immunoblot analysis of nuclear p50, p65, c-Rel and Oct-1 (loading control) in peripheral blood IgD+ B cells cultured for 6 h as described in a. Below lanes, quantification of relevant bands after normalization to actin; control value (far left lane in each) is arbitrarily set as 1. (l) Electrophoretic mobility-shift assay of nuclear NF-kB–Ig3 DNA complexes and Oct-1–DNA complexes (loading control) from peripheral blood IgD+ B cells cultured for 6 h as described in a. NF-kB-containing complexes were identified by preincubation of nuclear proteins from stimulated B cells with cold (unlabeled) kB3-Ig3 probe or antibody to p50, p65 or c-Rel before the addition of labeled kB3-Ig3 probe. Arrowheads indicate complexes supershifted or inhibited by the antibody. Data summarize three experiments (a–j; error bars, s.d.; *, P o 0.05) or are one of three experiments yielding similar results (k,l).

with DCs and B cells through a TLR-inducible SLPI-regulated signaling program (Supplementary Fig. 10 online). Such a pathway would ultimately elicit the production of polyreactive IgG and IgA antibodies targeting the microbial product(s) initially sensed by the ECs. DISCUSSION We have reported here that ECs lining tonsillar crypts formed pockets colonized by B cells expressing AID, an enzyme associated with ongoing class switching. ECs released B cell–activating AID-inducing factors, including BAFF and IL-10, after sensing viral RNA through TLR3. The resultant class switching caused the production of broadly reactive IgG and IgA antibodies, including antibodies to viral antigens. EC-induced class switching was enhanced by TSLP, an epithelial IL-7like cytokine that enhanced BAFF production by myeloid DCs, and was restrained by SLPI, an epithelial homeostatic protein that inhibited BAFF signaling in B cells. Our data indicate that ECs function as mucosal ‘guardians’ orchestrating frontline class switching through a TLR-inducible, SLPI-regulated signaling program. Antibodies are critical for host defense at mucosal sites of entry. Consistent with that, secretions bathing respiratory, gastrointestinal and genitourinary surfaces contain IgM as well as class-switched IgA and, to a smaller extent, IgG antibodies33. These immunoglobulins are released in the subepithelial environment by plasma cells originating from B cell precursors lodged in mucosal lymphoid follicles16,19,20. ECs use basolaterally expressed polymeric immunoglobulin and neonatal Fc receptors to transcytose immunoglobulins onto the mucosal surface34–39. ECs further enhance mucosal antibody responses by recruiting immunogobulin-secreting plasma cells into the subepithelial environment via the chemokine CCL28 (refs. 24,25). By showing that ECs stimulated class switching in B cells, our findings indicate that the mucosal epithelium functions not only as a terminal effector site but also as an inducer of mucosal humoral immunity.

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Mucosal humoral responses, including class switching, are thought to require the activation of B cells by other immune cells. After sampling antigen in the lumen, subepithelial DCs activate CD4+ T cells, thereby inducing them to express CD40L and TH2 cytokines13–15,42. These stimuli induce antigen-selected follicular B cells to undergo class switching and induce the production of antibodies with high affinity for invading pathogens16,20,21. In some mucosal areas, such as the intestinal lamina propria, B cells can also undergo class switching by interacting with DCs expressing CD40Lrelated factors, such as BAFF27–29,31. This T cell–independent pathway would enable mucosal B cells to rapidly produce low-affinity and broadly reactive antibodies to commensal bacteria and quickly replicating pathogens, including viruses16,30,32. Our results have shown that the upper respiratory tract provides an intraepithelial niche for frontline class switching and outline a previously unknown epithelial pathway for the activation of mucosal B cells. We detected B cells expressing the CSR-inducing enzyme AID in epithelial pockets of tonsillar crypts, which constitute elective sites of antigen sampling and entry. Intraepithelial class switching would occur through a CD40-independent pathway, as mucosal ECs had high expression of BAFF. This expression probably resulted from in vivo exposure of ECs to microbial products, as ECs released BAFF and other class switch–inducing factors, including IL-10, after sensing viral RNA. Consistent with its strategic position in the upper respiratory tract, the tonsillar epithelium contained abundant TLR3, an innate viral RNA receptor. TLR3 was also detected in intraepithelial DCs and B cells, suggesting that intraepithelial class switching involves intimate interaction among ECs, DCs and B cells. ECs modulate mucosal adaptive immune responses by activating DCs through TSLP17,18,45. In the presence of that IL-7-like cytokine, DCs initiate TH2 responses characterized by the production of IL-4 and IL-10 (ref. 13,15), two cytokines promoting class switching and

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ARTICLES antibody production21. By showing that TSLP stimulated DCs to produce more BAFF, our results have identified a TH2-independent pathway for TSLP-mediated antibody production and indicate that TSLP amplifies EC-induced class switching by linking ECs with DCs and B cells. Consistent with that, TSLP-producing ECs of tonsillar crypts interacted not only with B cells but also with myeloid and plasmacytoid DCs expressing BAFF. Our findings indicate that viral RNA initiates intraepithelial class switching and antibody production because of its ability to activate a TLR3-dependent signaling program sequentially involving topographically contiguous ECs, DCs and B cells. This suggests that mucosal humoral immune responses depend on the ability of microbial products to link ECs with DCs and B cells through TLRs. Accordingly, viral RNA cooperated with TSLP or BAFF to upregulate TLR3 expression by DCs or B cells, respectively. As a result of that upregulation, viral RNA stimulated DCs to express more TSLPR and to produce more BAFF and IL-10 in response to TSLP. In addition, viral RNA stimulated B cells to undergo more class switching and antibody production in response to BAFF and IL-10. Viral RNA–induced B cell responses were further increased by engagement of the antigen receptor (BCR), again through a mechanism involving upregulation of TLR3. These observations collectively suggest that intraepithelial IgG and IgA class switching is governed by integrated immune ‘circuits’ linking ECs with DCs and B cells through TLRs. EC-induced class switching would provide immune protection by generating IgG and IgA antibodies to multiple antigens, including the microbe originally sensed by the ECs. Consistent with this, ECs exposed to viral RNA stimulated B cells to produce IgG antibodies targeting viral antigens as well as bacterial products, including LPS and phosphorylcholine. Of note, EC-induced IgG antibodies reacted to some self antigens, including double-stranded DNA. Thus, although it confers increased flexibility to the mucosal immune barrier, ECinduced class switching has the potential to generate pathogenic autoantibodies and therefore must be tightly regulated. Our findings suggest that such regulation is provided by SLPI, the EC-derived ‘alarm’ antiprotease endowed with powerful homeostatic functions10. In addition to providing an antiprotease shield, SLPI has a key homeostatic function, as it regulates EC growth and has antiviral, antibacterial and anti-inflammatory activities10,46,47. Extracellular SLPI enters macrophages and thereafter interferes with their ability to undergo NF-kB-dependent production of proinflammatory tumor necrosis factor in response to LPS48. Like macrophages, B cells exposed to exogenous SLPI, including intraepithelial B cells, contained SLPI in both the cytoplasm and nucleus. In those B cells, SLPI inhibited class switching by interfering with the NF-kB-dependent transcriptional activation of germline IH promoters and with the upregulation of AID induced by viral RNA, BAFF and IL-10. These effects were specific, as they were not associated with lower B cell survival, B cell proliferation or IgM production. By showing that SLPI is produced by ECs at a later time point than are BAFF and TSLP, our findings indicate that SLPI may function as a negative feedback protein restraining the intraepithelial production of potentially pathogenic IgG and IgA antibodies. In conclusion, our studies here have indicated that ECs lining tonsillar crypts form a self-contained niche that promotes frontline IgG and IgA class switching through a TLR-dependent, SLPI-regulated signaling program. Our results suggest that mucosal vaccines should activate ECs in addition to immune cells to effectively stimulate protective IgG and IgA responses. Further studies will be needed to ascertain why class switching is heavily biased toward IgA in certain mucosal areas, such as the gut16,19,20,33. We propose that such an IgA

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bias stems from the ability of intestinal ECs to express a specific set of class switch–regulating factors. METHODS Cells. Human B cells were negatively selected from peripheral blood and tonsillar mononuclear cells with a commercially available kit (Miltenyi Biotec). Buffy coats from healthy donors were purchased at the New York Blood Center and were used to isolate peripheral blood mononuclear cells. Tonsillar mononuclear cells were obtained from tissue specimens of patients undergoing tonsillectomy. The Institutional Review Board of Weill Medical College of Cornell University approved the use of tonsil specimens for this study, and patients provided informed consent. IgD+ B cells were magnetically sorted by incubation of total B cells with a biotinylated monoclonal antibody (mAb) to IgD (2032-08; Southern Biotechnologies) and streptavidin MicroBeads (Miltenyi Biotec), which are specifically designed to minimize BCR crosslinking. All sorting procedures were done on ice. In some experiments, B cells were negatively selected with a commercially available kit (Miltenyi Biotec). Positively and negatively selected IgD+ B cells were similar in size and phenotype and in their functional responses to BAFF or CD40L (data not shown). The line 2E2 is a subclone of CL-01, a human IgD+ B cell line that initiates germline immunoglobulin gene transcription after exposure to the appropriate stimuli, including CD40L, BAFF and cytokines50. Primary oral ECs are commercially available (MatTek Corporation) and were cultured in modified Keratinocyte Cell Medium as instructed by the manufacturer. Those cells lacked contaminating DCs expressing CD11c, CD123 and/or langerin (Supplementary Fig. 1). In addition, oral ECs lacked transcripts for the C-type lectin receptor DC-SIGN, CD3 and Ig-b, which are specifically expressed by DCs, T cells and B cells, respectively (data not shown). Primary epidermal ECs are commercially available (Cambrex Bio Science Walkersville) and were cultured in Keratinocyte Cell Medium as instructed by the manufacturer. Intestinal Caco-2 ECs were cultured with RPMI 1640 medium. Myeloid DCs were obtained from peripheral blood monocytes. Monocytes were sorted with a biotinylated mAb to CD14 (UCHM; Serotec) and streptavidin MicroBeads and were then cultured for 6 d in RPMI 1640 medium (Invitrogen) supplemented with 5% human AB serum (Sigma), 1,000 U/ml of granulocyte-monocyte colony-stimulating factor (Berlex Laboratories) and 1,000 U/ml of IL-4 (R&D Systems). Every 2 d, 400 ml of medium was removed from each well and was replaced by 500 ml of fresh medium with the appropriate cytokines. After 6 d, more than 95% of the cells in culture expressed DC-specific antigens, including CD11c, DEC-205, and DC-SIGN (Supplementary Fig. 7). Cultures and reagents. Cells were cultured in complete RPMI medium supplemented with 10% (volume/volume) bovine serum. Reagents were used at the following concentrations: recombinant human BAFF (Alexis Biochemicals), 250 ng/ml; IL-4, 200 U/ml; IL-10 (Schering-Plough), 50 ng/ml; IFN-a (Sigma), 200 ng/ml; soluble trimeric CD40L (Immunex), 250 ng/ml; TSLP, 30 ng/ml; and SLPI (R&D Systems), 0.5 mg/ml. B cells were preincubated for 1–2 h with SLPI before the addition of other stimuli. Stimuli were used at the following concentrations unless otherwise indicated: polyclonal antibody to the m-chain of immunoglobulins (H15100; Caltag Laboratories), 2 mg/ml; poly(I:C), 10 mg/ml; PGN (InvivoGen), 20 mg/ml; LPS (Sigma), 10 mg/ml; and CpG ODN (Operon Technologies), 10 mg/ml. ECs (2 104) and B cells (1.0 106) with or without DCs (0.2 105) were seeded in the upper and lower chambers, respectively, of a 3.0-mm PET membrane transwell (BD PharMingen). BAFF was blocked with 30 mg/ml of BCMA-Ig (Ancell). TSLP was blocked with 30 mg/ml of blocking sheep polyclonal anti-TSLP (AF1398; R&D Systems). Mouse MOPC21 IgG1 and sheep immunoglobulins (Sigma) with irrelevant binding activity were used at a concentration of 30 mg/ml as controls for BCMA-Ig and antibody to TSLP (anti-TSLP), respectively. Flow cytometry. Monocytes and monocyte-derived myeloid DCs were stained with fluorescein-, phycoerythrin- or allophycocyanin-conjugated mAb 1D6 to BAFF, mAb eB-h209 to DC-SIGN (eBioscience), mAb HL3 to CD11c, mAb MoP9 to CD14, mAb 3/23 to CD40, mAb 16-10A1 to CD80, mAb HB-15e to CD83, mAb GL-1 to CD86, mAb M5-115 to HLA-II, mAb MG38 to DEC-205 and mAb 19.2 to the mannose receptor (BD PharMingen). At least 1 104 viable cells were acquired with a FACSCalibur analyzer (BD PharMingen). Histogram shifts were analyzed with the Kolmogorov-Smirnov test included

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with CellQuest software (BD PharMingen) and were considered statistically significant at P values of less than 0.001. Enzyme-linked immunsorbent assay (ELISA). Total IgG, IgA and IgM antibodies as well as BAFF and IL-10 were detected by standard ELISA as reported29,50. IgG and IgM antibodies to LPS and phosphorylcholine were detected in microplates coated with 20 mg/ml of LPS (Sigma) and 10 mg/ml of phosphorylcholine chloride (Sigma), respectively, in carbonate-bicarbonate buffer. IgG and IgM antibodies to double-stranded DNA were detected in microplates sequentially coated with 2 mg/ml of protamine sulfate (Sigma) in carbonate-bicarbonate buffer and 10 mg/ml of double-stranded DNA (Sigma) in PBS. IgG antibodies to influenza were detected in microplates coated with 2 mg/ml of inactivated H1N1 influenza A virus (American Type Culture Collection) in carbonate-bicarbonate buffer. Soluble TSLP was detected by incubation of microplates with primary sheep polyclonal anti-TSLP (AF1398; R&D Systems). The appropriate biotin- or peroxidase-conjugated secondary polyclonal antibodies or peroxidase-conjugated streptavidin and the substrate TMB (3,3¢,5,5¢-tetramethylbenzidine; Kirkegaard and Perry) were added in sequential steps. Readings were made at 450 nm. B cell survival assay and proliferation assays. B cell survival was evaluated with the Annexin V-FITC Apoptosis Detection Kit (Calbiochem) as instructed by the manufacturer. For measurement of B cell proliferation, 1 104 cells per 200 ml were seeded in 96-well plates and were pulsed with 1 mCi [3H]thymidine on day 4 of culture. After 18 h, cells were collected for measurement of [3H]thymidine uptake. Immunohistochemistry. Tonsil tissue samples were obtained from donors with tonsillitis; skin and intestinal tissue samples were obtained from donors with atopic dermatitis and from healthy tissue of colon carcinoma, respectively. Paraformaldehyde-fixed frozen tissue sections 5 mm in thickness were stained with various combinations of the following primary antibodies to human antigens: fluorescein-conjugated mouse mAb IA6-2 to IgD (BD PharMingen); biotin-conjugated goat F(ab¢)2 polyclonal antibody to immunoglobulins (201008), IgD (2032-08) or IgG (2042-08; Southern Biotechnologies); biotin-conjugated mouse mAb 3.9 to CD11c (Ancell); biotin-conjugated mouse mAb 6H6 to CD123 (eBioscience); unconjugated rat mAb 5B11 to CD123 (Serotec); unconjugated mouse mAb A-11 to Pax5 (Santa Cruz); unconjugated rabbit polyclonal antibody H-240 to ‘pan-cytokeratin’ (Santa Cruz); unconjugated rat antibody EK2-5G9 to AID (Ascenion); biotin-conjugated mouse mAb 1D6 to BAFF (eBioscience); unconjugated rabbit polyclonal anti-BAFF (07-167; Upstate Biotechnologies); biotin-conjugated mouse mAb TLR3.7 to TLR3 and mouse mAb 5G5 to TLR9 (HyCult Biotechnology); unconjugated mouse mAb DCGM4 to langerin (Immunotech); and biotin-conjugated sheep polyclonal anti-TSLP (BAF1398; R&D Systems). Unconjugated rabbit polyclonal anti-SLPI was obtained as described46,47. Control primary antibodies with irrelevant binding activity included fluorescein-conjugated, biotin-conjugated or unconjugated mouse IgG1 mAb, unconjugated rat IgG2a mAb and IgG2b mAb, biotinconjugated goat F(ab¢)2 polyclonal antibody, unconjugated rabbit polyclonal antibody and biotin-conjugated sheep polyclonal antibody (negative control staining, Supplementary Fig. 1). Slides were incubated with the following secondary reagents: indodicarbocyanine-conjugated polyclonal anti-mouse, rhodamine-conjugated polyclonal anti-mouse (Jackson ImmunoResearch Laboratories), Alexa Fluor 546–Alexa Fluor 488–conjugated polyclonal anti-goat, Alexa Fluor 647–conjugated polyclonal anti-rabbit and cyanine 3–cyanine 5–conjugated or rhodamine-conjugated streptavidin (Molecular Probes). Nuclei were visualized with DAPI (4¢,6-diamidine-2¢-phenylindole dihydrochloride; Boehringer Mannheim). Slow Fade reagent (Molecular Probes) was applied to slides, followed by analysis with a Zeiss Axioplan 2 microscope (Atto Instruments). In situ RNA hybridization. Full-length AICDA cDNA under control of the SP6 promoter was used as template for the transcription of an antisense RNA probe with SP6 RNA polymerase and biotin-labeled UTP. Frozen tonsil sections were fixed with paraformaldehyde and then were made permeable with 0.2% (weight/volume) Triton-X100 in PBS. The RNA probe (2 mg) was incubated with the slide for 36 h at 37 1C in Hybridization Buffer (Sigma). Slides were washed for three times with 2 sodium saline citrate solution and once with PBS-Tween. RNase (1% (weight/volume) in PBS) was then applied to slide for

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5 min at 37 1C. After an additional wash with PBS-Tween, slides were incubated for 30 min with streptavidin-indocarbocyanine and DAPI. Slow Fade reagent (Molecular Probes) was applied to slides, followed by analysis with a Zeiss Axioplan 2 microscope (Atto Instruments). RT-PCR and quantitative real-time RT-PCR. Total RNA was prepared with TRIzol (Invitrogen); cDNA was synthesized from total RNA as described29,50. TNFSF13B transcripts and ACTB transcripts were amplified by RT-PCR for 25 cycles as reported29,50. Real-time RT-PCR analyses were done in triplicate on the ABI PRISM 7900HT Sequence Detection System with the SYBR Green PCR kit as instructed by the manufacturer (Applied Biosystems). The amount of mRNA was normalized to the amount of ACTB mRNA. The generation of amplification products of only the correct size was confirmed by dissociation curves and agarose gel electrophoresis. The relative expression (RE) of a specific gene was calculated according to the equation REn ¼ 2–(DCtn – DCt1), where DCt (change in cycle threshold) is the cycle threshold of the test gene minus the cycle threshold of ACTB, n is a specific sample and 1 is the sample with the lowest expression. The following primer pairs were used: ACTB forward (5¢-GGATGCAGAAGGAGATCACT-3¢) and reverse (5¢-CGATCCACACGGAG TACTTG-3¢); AICDA forward (5¢-AGAGGCGTGACAGTGCTACA-3¢) and reverse (5¢-TGTAGCGGAGGAAGAGCAAT-3¢); Ig1/2-Cm forward (5¢-GGGC TTCCAAGCCAACAGGGCAGGACA-3¢) and reverse (5¢-AGACGAGGGGG AAAAGGGTT-3¢); Ig3-Cm forward (5¢-GCCATGGGGTGATGCCAGGATGGG CAT-3¢) and reverse (5¢-AGACGAGGGGGAAAAGGGTT-3¢); Ia1/2-Cm forward (5¢-CAGCAGCCCTCTTGGCAGGCAGCCAG-3¢) and reverse (5¢-AGACGA GGGGGAAAAGGGTT-3¢); Im-Cm forward (5¢-GTGATTAAGGAGAAACACTT TGAT-3¢) and reverse (5¢-AGACGAGGGGGAAAAGGGTT-3¢); Ig1-Cg1 forward (5¢-GACCTGAGCTCAGGAGGCAGCAGAGACC-3¢) and reverse (5¢-GA AGACCGATGGGCCCTTGGTGGA-3¢); Ig3-Cg3 forward (5¢-GCCATGGGGT GATGCCAGGATGGGCAT-3¢) and reverse (5¢-GAAGACCGATGGGCCCTT GGTGGA-3¢); SLPI forward (5¢-CCTGGATCCTGTTGACACCC-3¢) and reverse (5¢-CACTTCCCAGGCTTCCTCCT-3¢); TSLP forward (5¢-CCCAGGCTATTC GGAAACTCAG-3¢) and reverse (5¢-CGCCACAATCCTTGTAATTGTG-3¢); CRLF2 forward (5¢-TGGATCACAGACACCCAGAA-3¢) and reverse (5¢-TCTTG GCCAACTGGACTACC-3¢); TLR3 forward (5¢-TGACTGAACTCCATCTCATG TCC-3) and reverse (5¢-CCATTATGAGACAGATCTAATGTG-3¢); TLR9 forward (5¢-ACAACAACATCCACAGCCAAGTGTC-3¢) and reverse (5¢-AAGGC CAGGTAATTGTCACGGAG-3¢); and TNFSF13B forward ((5¢-ACCGCGGGA CTGAAAATCT-3¢) and reverse (5¢-CACGCTTATTTCTGCTGTTCTGA-3¢. RNA interference and nucleofection. ECs (1.5 106) were resuspended in 100 ml of Human Keratinocyte Nucleofector Solution (Amaxa) in the presence of pKD-BAFF-V2 plasmid expressing BAFF siRNA (5¢-AAGAAGGAGATGAACTC CAACTTCAAGAGAGTTGGAGTTCATCTCCTTCTTTTG-3¢) or pKD-NegConV1 plasmid expressing control siRNA (5¢-AAAGTCATCGACTAGCCTTACTT CAAGAGAGTTAAGGCCTAGTCGATGACTTTTTTG-3¢) (Upstate Biotechnologies). Plasmids were transfected by nucleofection with a Nucleofector device (Amaxa) with the program T-24. Expression of BAFF and actin by ‘nucleofected’ cells was evaluated after 72 h by RT-PCR and immunoblot analysis. Luciferase reporter assays. IgD+ 2E2 B cells (20 106 cells/ml), a subclone of the CL-01 cell line50, were transfected by electroporation with 40 ml of a solution of plasmid DNA and Tris-EDTA, pH 8.0, containing 10 mg Ig3-Luc or NF-kB(2)-Luc reporter plasmid expressing firefly luciferase, and 200 ng control pRL-TK reporter plasmid expressing renilla luciferase under control of the thymidine kinase promoter (Promega). Firefly and renilla luciferase activity was measured after 48 h with the Dual-Luciferase Assay System (Promega). The luciferase activity of the Ig3-Luc and NF-kB(2)-Luc reporter plasmids was normalized to that of the cotransfected pRL-TK control plasmid and is expressed as ‘fold induction’ (normalized luciferase activity of extracts from stimulated B cells divided by that of unstimulated B cells). Immunoblot analysis. Equal amounts of cytoplasmic or nuclear proteins were separated by 10% SDS-PAGE and were transferred onto nylon membranes (BioRad). After being blocked, membranes were probed with primary polyclonal or monoclonal antibodies to p50 (C-19), p65 (C-20), c-Rel (B-6), Pax5 (C-20), Oct-1 (12F11), actin (I-19; Santa Cruz Biotechnologies), BAFF (07-167; Upstate Biotechnology) or IkBa phosphorylated at Ser2 and Ser36 (5A5; Cell

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Signaling Technology). Membranes were then washed and were incubated with the appropriate peroxidase-conjugated secondary polyclonal antibody (Santa Cruz Biotechnologies). Proteins were detected with an enhanced chemiluminescence detection system (Amersham) and signal intensity was quantified with Quant1 software (Bio-Rad Laboratories). Electrophoretic mobility-shift assay and supershift assay. An oligonucleotide encompassing a kB3 site in the evolutionarily conserved sequence of the human Ig3 promoter was labeled with [a-32P]ATP and was used at approximately 30,000 c.p.m. in each electrophoretic mobility-shift assay. Reaction samples were prepared as described29,50 and were separated by 5% nondenaturing PAGE. The composition of DNA-bound protein complexes was determined by incubation of the reaction mixture with 1 mg polyclonal antibody to p65 (C-20), c-Rel (B-6) or p50 (C-19; Santa Cruz) before the addition of radiolabeled probe. Statistical analysis. For immunoglobulin secretion, proliferation, survival and reporter assays, values were calculated as mean standard deviation for at least three separate experiments done in triplicate. The significance of differences between experimental variables was determined with the paired Student’s t-test. Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTS We thank C. Nathan (Weill Medical College of Cornell University) and M. Rescigno (European Institute of Oncology) for reagents and discussions. Supported by the National Institutes of Health (AI057653 to A.C.; and T32 AI07621, supporting W.X.). AUTHOR CONTRIBUTIONS W.X. designed and did research and analyzed data; B.H., M.S. and M.D. did research; A. Chiu provided tissue samples and discussed data; A. Chadburn and D.M.K. provided tissue samples; A.D. provided reagents, discussed data and edited the paper; P.A.S. analyzed and discussed data; and A. Cerutti designed research, analyzed data and wrote the paper. COMPETING INTERESTS STATEMENT The authors declare that they have no competing financial interests. Published online at http://www.nature.com/natureimmunology/ Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions

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