Chemokine, CXC Chemokine Ligand 13 Induction of the B Cell-Activating

Home , CCL16, Chemokine, CXCL13, CXCL3, CXCL6

The Journal of Immunology

Distinct Transcriptional Programs Activated by Interleukin-10 with or without Lipopolysaccharide in Dendritic Cells: Induction of the B Cell-Activating Chemokine, CXC Chemokine Ligand 131

Patrick Perrier,* Fernando O. Martinez,† Massimo Locati,† Giancarlo Bianchi,* Manuela Nebuloni,‡ Gianluca Vago,‡ Flavia Bazzoni,§ Silvano Sozzani,*¶ Paola Allavena,* and Alberto Mantovani2*†

To understand the modulation of dendritic cell (DC) function by IL-10, gene expression profiling was performed by using Af- fymetrix technology (Santa Clara, CA) in human monocyte-derived DC treated with IL-10, alone or in combination with LPS. The modulation of selected genes was validated by real-time PCR, Northern blot, and protein production. IL-10 regulated in DC the expression of a limited number of genes, including IL-7, the receptors for transferrin and vitamin D3, structural matrix proteins, and signal transduction elements. The combined treatment with LPS plus IL-10 modulated a number of genes comparable to LPS alone, but the expression profiles were distinct. As expected, IL-10 suppressed the expression of several LPS-inducible proinflammatory molecules. Among genes uniquely modulated by the concomitant treatment with LPS plus IL-10, phosphatidylinositol 3-kinase ␥ was down-regulated while the suppressor of cytokine signaling 3, signaling lymphocytic activation molecule, regulator of G protein signaling 16, and the chemokine, CXC chemokine ligand (CXCL) 13, were up-regulated. Overall, four distinct transcriptional programs were identified, related to: 1) control of immunity and inflammation; 2) tuning of cytokine receptor and G protein-coupled receptor signaling; 3) remodeling of extracellular matrix; and 4) B cell function and lymphoid tissue neogenesis. Among the latter genes, we further demonstrate that IL-10 synergizes with TLR ligands for the production of functionally active B cell-attracting chemokine, CXCL13, in both myeloid and plasmacytoid DC. This novel finding reveals that IL-10 sustains humoral immunity by inducing the production in APCs of the chemokine, CXCL13, which amplifies B cell recruitment and promotes lymphoid tissue neogenesis. The Journal of Immunology, 2004, 172: 7031–7042.

endritic cells (DC)3 are powerful APCs specialized in eloid precursors, of which plasmacytoid DC (pDC) and Langer- the priming of resting T cells and in the initiation of the hans cells are the major components (2–6). D immune response (1, 2). DC originate from bone mar- At their immature stage, DC are sparse in peripheral tissues and row and circulating precursors and can be distinguished in at least stand guard for the encounter of an Ag. DC express a rich reper- two subsets: myeloid-derived DC and DC derived from nonmy- toire of surface receptors such as Toll-like (TLR) and pattern recognition receptors (PRR) which allow the recognition of pathogen molecules (7–10). The engagement of TLR and PRR on DC ini- tiates a cascade of signaling events leading to the secretion of *Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; †Centro di Eccellenza inflammatory and immuno-modulatory factors mediating protec- per l’Innovazione Diagnostica e Terapeutica, Institute of General Pathology, Univer- tive immunity (7). For instance, activation of DC through engage- sity of Milan, Milan, Italy; ‡Institute of Pathology, University of Milan, Ospedale Luigi Sacco, Milan, Italy; §Department of Pathology, University of Verona, Verona, ment of TLR4 by LPS induces up-regulation of costimulatory Italy; and ¶Department of Biotechnology and Biomedical Sciences, Section of Gen- and MHC molecules, of maturation markers such as CD83, DC- eral Pathology and Immunology, University of Brescia, Brescia, Italy lysosome-associated membrane protein, and CCR7, increased Received for publication December 12, 2003. Accepted for publication March secretion of cytokines and chemokines, and potent APC function 12, 2004. (1, 2, 11). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance The transition of resting to mature DC is a critical step for the with 18 U.S.C. Section 1734 solely to indicate this fact. optimal initiation of immunity. Several studies have provided 1 This work was supported by grants from the European Commission and MIUR- evidence that activation/maturation of DC is a highly modulable FIRB Grant RBNE01Y3N3. P.P. was supported by Grant 3235-57619.99 of the Swiss process that can be accelerated or inhibited by several biological National Science Foundation. Transcriptional profiling was supported by Associazi- one Italiana Ricerca sul Cancro. factors. One such critical factor is IL-10. IL-10 inhibits the differ- 2 Address correspondence and reprint requests to Dr. Alberto Mantovani, Istituto di entiation of new DC from monocytic precursors and blocks DC Ricerche Farmacologiche Mario Negri, Via Eritrea 62, I-20157 Milan, Italy. E-mail maturation induced by LPS, as well as functional activities asso- address: [email protected] ciated with their mature state (6, 12–17). 3 Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; PRR, IL-10 is a multifunctional cytokine whose general effects are pattern recognition receptor; CXCL, CXC chemokine ligand; GPCR, G protein-coupled receptor; SOCS3, suppressor of cytokine signaling 3; HPF, high power field; aimed to limit the inflammatory response and prevent tissue dam- PTX3, pentraxin 3; CCL, CC chemokine ligand; PI3K␥, phosphatidylinositol 3-ki- age. This is achieved by down-regulating the expression of inflam- nase ␥; PTPN, protein-tyrosine phosphatase; SLAM, signaling lymphocytic-activating molecule; RGS16, regulator of G protein signaling 16; pDC, plasmacytoid DC; matory cytokines/chemokines, and inhibiting effector functions of JAK, Janus kinase; ODN, oligonucleotide. T cells and mononuclear phagocytes (17). In contrast, IL-10, by

suppressing Th1-polarized T cell responses and by inducing the independent RNA pools of three donors each were labeled, processed, and differentiation of suppressor/T regulatory cells, can also inhibit the independently hybridized on human genome U95Av2 arrays (HG- development of protective immunity. Thus, this cytokine plays a U95Av2; Affymetrix, Santa Clara, CA) containing 12,500 full-length genes, as previously described (26, 27). Scanned images were processed critical role in the balance between immune protection and using Microarray Suite 5.0 (Affymetrix), and raw data were analyzed using pathology. Data Mining Tool 3.0 (Affymetrix). The transcriptional profiles obtained in However, the effect of IL-10 in mononuclear phagocytes is not this first set of DC preparations were confirmed in a subsequent set of three purely inhibitory. In fact, IL-10 increases the differentiation of donors. Modulated genes were defined according to the following criteria: detection of p Յ 0.005; 100 Յ expression signal Յ 9000; change confi- monocytes into macrophages and enhances the endocytic ability of dence interval Ͼ99%; fold change in expression Ͼ2 and difference of DC (13, 18, 19) and induces the production of selected chemokines expression Ͼ150 intensity arbitrary units. Only those genes whose mod- such as CC chemokine ligand (CCL) 18 (20) and CCL16 (21). ulation was consistently observed in each of the three donors pools were IL-10 also up-regulates expression of selected chemokine recep- considered in our analysis and consequently presented in this work (Figs. tors (22). Moreover, DC exposed to a combination of IL-10 and 1 and 2, Tables II and III). Among the genes modulated in DC after combined treatment with LPS the prototypic inflammatory signal, LPS, retain at their surface plus IL-10, we were interested in the genes falling into the following cat- high levels of inflammatory chemokine receptors, which do not egories: 1) synergistically increased compared with single treatments, 2) elicit migration and act as decoys and scavengers for inflammatory synergistically decreased compared with single treatments, 3) increased by chemokines (16, 23). LPS and counterregulated by the addition of IL-10, and 4) decreased by LPS and counterregulated by the addition of IL-10. In an effort to explore the complexity of the biological role of Genes which were synergistically increased after LPS plus IL-10 com- IL-10 on DC, we conducted a genome-wide analysis of gene ex- pared with LPS or IL-10 alone had to meet the following three criteria: 1) pression by human myeloid DC upon exposure to IL-10. Further- intensity signal of LPS plus IL-10 Ͼ 2 times average of the intensity more, as during an inflammatory process, IL-10 is generated, and signals of untreated, LPS alone, and IL-10 alone; 2) intensity signal (av- Ͼ therefore, both proinflammatory and anti-inflammatory signals are erage centered) of LPS plus IL-10 2 times maximum intensity signal (average centered) among untreated, LPS alone, and IL-10 alone; and 3) locally present, we analyzed also the transcriptome of DC con- intensity signal (average centered) of LPS plus IL-10 Ͼ 100 plus average comitantly treated with LPS and IL-10. Under these conditions, it of the intensity signals (average centered) of untreated, LPS alone, and is well known that several genes up-regulated by LPS are coun- IL-10 alone. Genes which were synergistically decreased after LPS plus terregulated by IL-10. In this paper, we focused our attention on IL-10 compared with LPS or IL-10 alone had to meet the following three criteria: 1) intensity signal of LPS plus IL-10 Ͻ 0.5 times average of the those genes whose induction with LPS plus IL-10 treatment sig- intensity signals of untreated, LPS alone, and IL-10 alone; 2) intensity nificantly differed from treatments with LPS or IL-10 alone. signal (average centered) of LPS plus IL-10 Ͻ 2 times minimum intensity In the complex interplay mediated by IL-10 with or without signal (average centered) among untreated, LPS alone, and IL-10 alone; LPS, we identified at least four distinct functional programs: 1) and 3) intensity signal (average centered) of LPS plus IL-10 Ͻ (Average of genes related to the control of specific immunity and inflammation, the intensity signals (average centered) of untreated, LPS alone and IL-10 alone) Ϫ 100. 2) genes involved in tissue remodeling, 3) genes mediating the The average centered signal can be mathematically defined as: original tuning of cytokine receptor and G protein-coupled receptor signal of the considered treatment Ϫ average of the original signals of (GPCR) signaling, and 4) genes promoting B cell development/ (“untreated”, “LPS”, “IL-10”, and “LPS plus IL-10”). function and lymphoid tissue neogenesis. Finally, among genes Genes in which the modulation triggered by LPS was inhibited by the addition of IL-10 had to meet the following two criteria (genes whose related to B cell biology, we further studied the expression of the expression is increased by LPS): 1) intensity signal of LPS/Intensity signal B cell attracting chemokine, CXC chemokine ligand (CXCL) 13, of LPS plus IL-10 Ͼ and 2) Intensity signal of LPS Ϫ Intensity signal of and demonstrated that IL-10 strongly augments the LPS-induced LPS plus IL-10 Ͼ 100. The criteria used for those genes whose expression production of functionally active CXCL13 by DC. This novel find- is decreased by LPS were: 1) intensity signal of LPS/intensity signal of Ͻ Ϫ ing reveals that IL-10 sustains humoral immunity by inducing the LPS plus IL-10 0.5 and 2) intensity signal of LPS plus IL-10 intensity signal of LPS Ͼ 100. production, in APCs, of a chemokine which amplifies B cell recruitment and promotes lymphoid tissue neogenesis. Real-time PCR and Northern blot analysis DC of three healthy donors, other than those used for transcriptional pro- Materials and Methods filing, were exposed to LPS and IL-10 and checked for chemokine receptor Preparation and treatment of DC expression and migration (16) as a quality control. Total RNA was ob- ␮ Cells were obtained as described (24, 25). Briefly, 95% pure monocytes (as tained as described (26). Starting with 1 g of total RNA, the synthesis of assessed by CD14 analysis by flow cytometry) were obtained by using cDNA was performed by using the TaqMan Reverse Transcription Re- agents (Applied Biosystems, Foster City, CA). PCR was done using SYBR CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). DC ϫ were obtained by culturing monocytes for 6 days at 1 ϫ 106 cells/ml in Green PCR Core Reagents mix (Applied Biosystems) containing 1 RPMI 1640 (Biochrom, Berlin, Germany) with 10% FBS (HyClone, Lo- SYBR Green PCR buffer; 3 mM MgCl2; 100 mM dATP, dCTP, and dGTP; gan, UT) containing 50 ng/ml GM-CSF (Novartis Pharmaceuticals, Basel, 200 mM dUTP; 0.025 U/ml AmpliTaq Gold DNA polymerase; 0.01 U/ml Switzerland) and 10 ng/ml IL-13 (a gift from Dr. A. Minty, Sanofi Elf Bio AmpErase UNG. Gene-specific forward and reverse primers were designed Recherches, Labe`ge, France). Myeloid DC and pDC were obtained from using the Primer Express software (Applied Biosystems) and were pro- buffy coats of healthy donors using the CD1c (BDCA-1) dendritic cell vided by Invitrogen (Carlsbad, CA). They were used at 2 pmol/ml in the isolation and the BDCA-4 cell isolation kits (Miltenyi Biotec). LPS (Esch- reaction. Sequences are presented in Table I. erichia coli 055:B5; Sigma-Aldrich, St. Louis, MO) was used at 10 ng/ml; The reaction conditions were as follows: 2 min at 50°C (1 cycle), 10 min IL-10 (Schering-Plough, Kenilworth, NJ) was used at 50 ng/ml; and CpG at 95°C (1 cycle), 15 s at 95°C, and 1 min at 60°C (40 cycles). Gene- oligonucleotides (ODN) motif GTCGTT (2006) was provided by Invitro- specific PCR products were continuously measured by means of an ABI gen Life Technologies (Rockville, MD) and used at 2 ␮g/ml. PRISM 5700 detection system (PerkinElmer/Cetus, Norwalk, CT). Sam- ples were normalized using the housekeeping gene, GAPDH. Triplicates cRNA preparation, hybridization, and microarrays analysis were performed for each experimental point. Results are shown as a fold modulation over control. For transcriptional profile analysis, DC of six healthy donors were pre- In one case (SOCS3), the microarray analysis was validated by Northern pared, resuspended at 1 ϫ 106 cells/ml in RPMI 1640 supplemented with blot after 2- and 8-h exposure to LPS, IL-10, and LPS plus IL-10. Total 10% FBS, and stimulated with IL-10, LPS, or a combination of both stim- RNA was extracted by the TRIzol method, blotted, and hybridized as de- uli for 2 and 8 h. As quality control of the stimulation, phenotype analysis scribed (22). A specific SOCS3 probe was obtained by using the hSOCS and functional activity of the cells were evaluated (16). Total RNA was MultiProbe Template Set (BD PharMingen, San Diego, CA), following the obtained as described (26), quantified, and normalized among donors ac- instructions of the manufacturer, and labeled by Megaprime DNA labeling cording to GAPDH levels, as determined by real-time PCR analysis. Two system (Amersham, Buckinghamshire, U.K.) with [␣-32P]dCTP (3000 Ci/ The Journal of Immunology 7033

Table I. Primers used for real-time PCR validation of the roTech, Rocky Hill, NJ) and DC supernatants were used as chemotactic transcriptional profiles factors. To inhibit the migration in response to CXCL13, a specific mAb (clone 53610.11, mouse IgG1; R&D Systems) was placed at 20 ␮g/ml in the lower compartment of the chemotactic chamber. An isotype-matched Gene Sequence murine mAb was used as negative control. Results presented are numbers CXCL13 Forward 5Ј-TCTCTGCTTCTCATGCTGCTG-3Ј of cells counted in 10 high power fields (HPF). CXCL12 (300 ng/ml) or Reverse 5Ј-TTCGATCAATGAAGCGTCTAGG-3Ј CXCL13 (10 ng/ml) were used as positive controls. IL-12B Forward 5Ј-GTCTTAGGCTCTGGCAAAACCC-3Ј Reverse 5Ј-AGCAGGAGCGAATGGCTTAGA-3Ј Statistical analysis Ј Ј Caspase 5 Forward 5 -ACCGCAACTGCCTCAGTCTAAA-3 Statistical analysis of the microarray experiment has been extensively ex- Ј Ј Reverse 5 -ATGCTGGAGAGTCTCTGACCCA-3 plained above. Data of real-time PCR, ELISA, and chemotaxis experiments Ј Ј CCR7 Forward 5 -TGCATCAGCATTGACCGCTA-3 are expressed as the mean values Ϯ SE. Significance was assessed by using Ј Ј Reverse 5 -TATCCAGATGCCCACACAGGA-3 the Student’s t test. For correlation analysis presented in Fig. 7A, R2 was Ј Ј RGS16 Forward 5 -TTTGCAGTGAGGCCCCTAAAG-3 calculated as the correlation coefficient of the linear regression and p value Ј Ј Reverse 5 -TGAGCCGCATCAAAGCATG-3 was determined by a Student’s paired t test. TUBB2 Forward 5Ј-ATCAGCAAGATCCGGGAAGAG-3Ј Reverse 5Ј-CCGTGTCTGACACCTTGGGT-3Ј NR4A3 Forward 5Ј-TCAGCCTTTTTGGAGCTGTT-3Ј Results Reverse 5Ј-TGAAGTCGATGCAGGACAAG-3Ј Distinct but overlapping transcriptional profiles of DC exposed CSPG2 Forward 5Ј-TTGGACTGATGGCAGCACACT-3Ј to LPS and IL-10 (versican) Reverse 5Ј-GGCCATTCTCATGCCAAATG-3Ј TNFRSF1B Forward 5Ј-GGCAAGTCCCTGACTCTCTG-3Ј Fig. 1 provides an overview of the transcriptional profile of DC Reverse 5Ј-CAGGGGGAGAAACAAACAAA-3Ј exposed to LPS, IL-10 or a combination of the two. In agreement INFB1 Forward 5Ј-CCTCCAAATTGCTCTCCTGTTG-3Ј with previous reports (30–32), LPS had a profound effect on the Ј Ј Reverse 5 -CAATTGCCACAGGAGCTTCTG-3 transcriptome expressed in human DC. By the stringent criteria SERPINB2 Forward 5Ј-TCCATTCATCCTTCCGCTCTC-3Ј Reverse 5Ј-GCTCGCAGACTTCTCACCAAAC-3Ј GAPDH Forward 5Ј-AGATCATCAGCAATGCCTCCTG-3Ј Reverse 5Ј-ATGGCATGGACTGTGGTCATG-3Ј

mmol; Amersham). Membranes were prehybridized at 42°C in Hybrisol (Oncor, Gaithersburg, MD) and hybridized overnight with 1 ϫ 106 cpm/ml 32P-labeled probe. Membranes were then washed 3 times with 2ϫ SSC (1ϫ SSC ϭ 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), at room temperature for 10 min, twice with 2ϫ SSC, 1% SDS at 60°C for 20 min, and then with 0.1ϫ SSC for 5 min, before being exposed to XAR-5 films (Kodak, Rochester, NY) using intensifier screens at Ϫ80°C. Protein analysis After a 6-h exposure to LPS, IL-10, and LPS plus IL-10, DC were stained with anti-signaling lymphocyte activation molecule (SLAM) mAb (clone HM2a-IPO3, mouse IgG2A; Advanced ImmunoChemical, Long Beach, CA) at 1/1000 dilution. An isotype-matched murine mAb was used as negative control. Ab binding was detected by using FITC-conjugated goat anti-mouse IgG 1/25 dilution (Southern Biotechnology Associates, Bir- mingham, AL) and samples were analyzed by flow cytometry. For Western blot analysis, preparation of cell lysates and immunoblot analysis were conducted as previously described (28) using an anti-SOCS3 mAb (Im- muno-Biological Laboratories, Tokyo, Japan) diluted at 5 ␮g/ml. Ab binding was detected by using HRP-conjugated anti-mouse IgG at 1/4000 dilution and revealed using the ECL system (Amersham Pharmacia Biotech, Arlington Heights, IL). To confirm equal protein loading per lane, filters were stripped and reprobed with Abs anti-p38 mitogen-activated protein kinase (New England Biolabs, Beverly, MA) diluted as recommended by the manufacturer. Supernatants of DC, resuspended at 1ϫ 106/ml in RPMI 1640 supplemented with 10% FBS and exposed for 18 h to LPS with or without IL-10, were tested for CXCL13 concentration by using the human BLC/BCA-1/CXCL13 DuoSet ELISA (R&D Systems, Minneapolis, MN). Pentraxin 3 (PTX3) was measured in supernatants of DC (1 ϫ 106/ml in RPMI, 0.2% BSA) by ELISA as previously described (29). In some experiments, DC were treated with LPS in the presence of anti-IL-10 mAb (2 ␮g/ml, mouse IgG2B, clone 23738; R&D Systems). An anti-CCR2 mAb (2 ␮g/ml mouse IgG2B, clone 48607; R&D Systems) was used as a negative control. Chemotaxis of B cells Freshly isolated B lymphocytes and the DHL-4 follicular lymphoma cell line were used. Fresh B lymphocytes were obtained from human tonsils by FIGURE 1. General description of the expression profiles obtained after using the B cell separation kit (Miltenyi Biotec), following the instructions treatment of DC with LPS, IL-10, and a combination of the two stimuli. A, of the manufacturer. Migration assays were conducted for 3 h using 48- well chambers (NeuroProbe, Cabin John, MD) with 8-␮m polyvinylpyr- Number of genes which are increased (open bars) or decreased (filled bars) rolidone-free filters coated with 10 ␮g/ml fibronectin, as previously de- after 2 and 8 h. B, Overlap between the set of genes regulated by LPS, scribed (16). B lymphocytes were preincubated in migration medium IL-10, and the combination of the two. Square areas are proportional to the (RPMI 1640, supplemented with 2.5% FBS) for 2 h and seeded (1.5 ϫ number of modulated genes. Only the 8-h time point is shown, but similar 105/well). Recombinant human CXCL13 (R&D Systems), CXCL12 (Pep- data were obtained at 2 h. 7034 IL-10-MODULATED TRANSCRIPTION IN DC

Table II. IL-10-regulated genesa

a Genes modulated by IL-10 in DC after 2 and 8 h. For comparison, the signals after LPS and LPSϩIL-10 are also shown. Green indicates an increased expression, and pink a decreased expression, compared to untreated cells. Only the modulations which fulﬁll the criteria presented in Materials and Methods are colored. Values represent the original signals of one experiment. All the genes shown are modulated in the same way in another independent experiment. The Journal of Immunology 7035

Table III. Interplay between LPS and IL-10 in the regulation of gene expressiona 7036 IL-10-MODULATED TRANSCRIPTION IN DC

Table III. (Continued)

a Genes modulated by the combined treatment LPSϩIL-10 in DC after 2 and 8 h. Color code: genes increased (light green) or decreased (pink) compared to untreated cells; genes synergistically increased (dark green) or synergistically decreased (red) by LPSϩIL-10 compared to single treatements; genes regulated by LPS and counter-regulated by IL-10 (blue). Only the modulations which fulfill the criteria presented in Materials and Methods are colored. Values represent the original signals of one experiment. All the genes shown are modulated in the same way in another independent experiment. used in the present study, after2hofexposure, LPS reproducibly from that activated by LPS, with limited overlap (Fig. 1B). For increased expression of 177 genes and decreased that of 216 genes. instance, at the 8-h time point, only 8 of the 19 IL-10-regulated Comparable numbers of modulated genes were observed at the 8-h genes were also induced by LPS. The overall transcriptional profile time point. In contrast, IL-10 regulated expression of a restricted of cells exposed to combined LPS plus IL-10 partially overlapped set of genes, ϳ10-fold lower than that modulated by LPS. With the with, yet was distinct from, that of cells treated with LPS alone. stringent criteria used in the present study, the number of genes For instance, at 8 h, overall 474 genes were positively or nega- augmented by IL-10 was 26 and 19 at 2 and 8 h, respectively, tively regulated by combined LPS and IL-10, compared with 531 whereas the decreased ones were 9 and 12 at 2 and 8 h, respec- for LPS and 31 for IL-10. For the 8-h time point, 76 genes induced tively. The transcriptional profile of IL-10-treated DC was distinct by IL-10 plus LPS were not stimulated by LPS or IL-10 alone. The Journal of Immunology 7037

Transcripts regulated by IL-10 with or without LPS Several studies have analyzed the transcriptional proﬁle of LPS- stimulated monocytes or DC (26, 30–32). The set of genes modulated by LPS in the present investigation is generally consistent with these previous reports and is available at http://www.marionegri.it/ proﬁle3. For instance, as expected, LPS induced expression of a number of chemokines (e.g., CCL1, CCL7, CCL19, CXCL3, CXCL6, and CXCL11) and cytokines (e.g., IL-12A, IL-12B, IFN-␥, and IFN-␤1). Table II lists genes induced or augmented by IL-10 alone, and by way of comparison, the effect of LPS and LPS plus IL-10 on the same transcripts. IL-10 inducible genes included a distinct set of cytokines/cytokine receptors: IL-7, IL-4R␣ subunit and pre-B colony enhancing factor. The latter is a cytokine enhancing the pro- liferative effect of stem cell factor and IL-7 on precursor B cells

(33). In light of the effect of IL-10 on B cells and in TH2-polarized responses, it is interesting to note that all these molecules are related to B cell differentiation and function. In addition, the set of IL-10 inducible genes included the long PTX3 and chondroitin sulfate proteoglycan 2 (versican), two molecules related to the extracellular matrix. IL-10 augmented the expression of the nuclear receptor for vitamin D3, a molecule with immunosuppressive activity. Among signaling molecules, IL-10 inhibited expression of phosphatidylinositol 3-kinase ␥ isoform (PI3K␥) and of mitogen- activated protein kinase kinase kinase 4 (MAP3K4), while it stimulated two protein-tyrosine phosphatases 1 and 2 (PTPN1 and PTPN2). SLAM transcript expression was increased by IL-10 after 2 h and the combination of LPS and IL-10 further increased transcript expression. Fig. 2 summarizes at a global level the interplay between LPS and IL-10 in the regulation of the gene expression in DC, while Table III shows the genes for which a significant interaction was observed between LPS and IL-10 in transcript regulation. As expected, IL-10 inhibited the LPS-induced augmented expression of a number of genes (19 and 21, at 2 and 8 h, respectively, Fig. 2A). The genes induced by LPS and counterregulated by IL-10 included cytokines (e.g., IL-12B and IFN-␤), chemokines (e.g., CCL1 and CXCL6) and the chemokine receptor CCR7. IL-10 also reverted the LPS-induced inhibition of 8 genes at 8 h (e.g., FZD2). In addition to counteracting the action of LPS, IL-10 had additive or synergistic effects on a substantial fraction of the transcriptome examined. IL-10 and LPS had additive positive effects on FIGURE 2. Interplay between LPS and IL-10 in the regulation of gene expression of 38 and 32 genes at 2 and 8 h, respectively, and expression. Upper panel, Number of genes whose expression is increased additive negative effects on two genes for each time point. In ad- or decreased by LPS in DC, and whose modulation is counterregulated dition, 22 and 14 genes not significantly affected by the individual under treatment by LPS plus IL-10. Lower panel, Number of genes which are synergistically increased or synergistically decreased after treatment by agents were induced by the combined exposure to IL-10 and LPS, LPS plus IL-10, compared with single treatments after 2 and 8 h. The after 2 and 8 h, respectively. The genes induced in an additive or window in the upper right corner indicates the expression behavior of synergistic way by combined treatment with LPS and IL-10 in- genes under single treatments (C (changed), modulated; NC (nonchanged), cluded the pre-B colony-enhancing factor, the B cell attractant nonmodulated). chemokine CXCL13, the regulator of G protein signaling (RGS16), SOCS3, PTPN1 and PTPN2, and the surface molecule, SLAM. Chondroitin sulfate proteoglycan 2 (versican) was also significantly superinduced. PI3K␥ was modulated by IL-10, and the ferred to as “superinduced genes”). As shown in Fig. 3A, by real- ␤ effect of IL-10 was not significantly modified by LPS (Table II). time PCR analysis expression of CCR7, IL-12B, IFN- 1, SER- PTX3 was stimulated by IL-10 and by LPS, although the syner- PINB2, TUBB2, and NR4A3 was augmented by exposure to LPS gistic effect was not scored significant (Table II). and inhibited by concomitant IL-10. For instance, expression of CCR7 was increased to 260.7 Ϯ 57.7 arbitrary units by LPS and suppressed to 54.1 Ϯ 11.5 arbitrary units by the addition of IL-10. Validation of the microarray transcriptional profile In contrast, caspase 5, CXCL13, RGS16, versican, and In an effort to validate the microarray transcriptional analysis, real- TNFRSF1B were induced by LPS and their expression was super- time PCR was performed on DC obtained from three individual induced by exposure to LPS and IL-10. RGS16 and versican were donors other than those investigated in the gene chip studies. We also induced by IL-10 alone. Moreover we took advantage of selected six genes whose induction by LPS was inhibited by con- availability of blotted RNA samples and a specific probe from comitant exposure to IL-10 (referred to as “counterregulated prior independent experiments to validate the regulation of SOCS3 genes”) and five superinduced by combined LPS and IL-10 (re- by Northern blot analysis. As shown in Fig. 3B, SOCS3 mRNA 7038 IL-10-MODULATED TRANSCRIPTION IN DC

FIGURE 3. Validation by real-time PCR and Northern blot analysis of transcriptional profiles. A, The modulation of the expression of SERPINB2, IFN-␤1 (IFN-B1), IL-12B (p40), ␤-tubulin (TUBB2), CCR7, NR4A3, TNFRSF1B, RGS-16, Versican, Caspase-5, and CXCL13 was measured by real-time PCR after treatment with LPS, IL-10, and LPS plus IL-10. The y-axis represents the mRNA expression in arbitrary units. Shown are mean Ϯ SE values of three independent -Signifi ,ء .experiments with healthy donors cantly different from LPS treatment (p Ͻ 0.05). #, Significantly different from LPS and IL-10 single treatments (p Ͻ 0.05). B, North- ern blot analysis of SOCS3 expression, representative of three experiments.

was induced by LPS and IL-10 and superinduced by a combination effect of IL-10 on human pDC. pDC express high levels of TLR9 of the two. Therefore, real-time PCR and Northern blot analysis on and low levels of TLR4. Therefore, a CpG oligonucleotide (ODN a set of 12 genes studied in three independent donors validated the motif GTCGTT (2006)) was used as agonist in these experiments. transcriptional profiling. In a series of three donors (Fig. 5B), the CpG ODN induced low levels (1.0 Ϯ 0.4 ng/ml) of CXCL13 in pDC. IL-10, inactive per Protein production: superinduction of CXCL13 se, dramatically increased CXCL13 production in pDC (7.1 Ϯ 1.8 A series of modulated genes, selected among the unexpected IL- ng/ml, p Ͻ 0.01). Freshly isolated blood myeloid DC behaved 10-inducible or -superinducible genes, were investigated at the like monocyte-derived DC when challenged with LPS and IL-10 protein level. As shown in Fig. 4, in agreement with microarray (Fig. 5B). analysis, IL-10 alone or in concert with LPS induced protein ex- These results indicated that indeed IL-10 costimulates CXCL13 pression of SOCS3 (Western blot). The up-regulation of PTX3 production in DC in concert with TLR engagement. As in some after IL-10 alone was also confirmed (ELISA). Increased expres- experiments, LPS alone induced a considerable release of sion of SLAM protein (flow cytometry) was observed only after CXCL13 (Fig. 5), we considered whether the LPS-induced endog- the combined treatment with LPS and IL-10. enous IL-10 could play a role in CXCL13 production. Therefore, Microarray and real-time PCR analysis unexpectedly revealed we measured IL-10 in the supernatants of DC from 12 donors who that IL-10 superinduces CXCL13 in human monocyte-derived DC had been stimulated with LPS and evaluated whether there was a (see above). Because IL-10 positively influences only a restricted correlation between endogenous production of IL-10 and set of (for instance CCL18 (20) and CCL16 (21)) it was important CXCL13. As shown in Fig. 6A, there was a significant ( p Ͻ 0.05), to assess the actual significance of this observation. CXCL13 was though far from strict (R2 ϭ 0.332) correlation between IL-10 measured by ELISA in a set of 12 donors. As shown in Fig. 5A, levels and CXCL13 levels in this series of 12 donors. To directly IL-10 alone never induced appreciable amounts of CXCL13 under assess the role of endogenous IL-10 in LPS induction of CXCL13, these conditions. LPS induced production of this chemokine (Ͼ0.6 a blocking mAb was used. In a series of three independent exper- ng/ml) in 5 of 12 donors. In both LPS-responsive and -nonrespon- iments, anti-IL-10 significantly reduced the LPS induction of sive DC, IL-10 significantly induced, in concert with LPS, or am- CXCL13 in DC, confirming the important costimulatory role of plified LPS-induced production of CXCL13. When freshly isolated IL-10 (Fig. 6B). monocytes were tested, LPS induced the release of a significant To test the functional activity of CXCL13 produced by DC amount of CXCL13 (1.5 Ϯ 0.2 ng/ml) in three independent ex- treated with LPS and IL-10, we performed chemotaxis assays us- periments (not shown). Surprisingly, in this cellular context, IL-10 ing supernatants from DC stimulated with IL-10 and LPS. Freshly did not synergize with LPS in the production of CXCL13. isolated tonsillary B lymphocytes significantly responded to DC are heterogeneous and pDC represent a phenotypically and CXCL13-containing supernatants but not to control supernatant. functionally distinct subset (1, 4, 34). We therefore examined the The highest migration was observed with 1/3 dilution (Ϸ0.5 ng/ml The Journal of Immunology 7039

FIGURE 4. Validation of transcriptional profiles by protein production after exposure to IL-10 alone or in concert with LPS. A, Up-regulation of SLAM/CD150 after 6-h incubation. The presented diagram is an average of three FACS experiments. B, Induction of SOCS3 after 18-h incubation. The Western blot shown here is representative of three experiments. p38 mitogen-activated protein kinase was used for normalization. C, Induction of PTX3 after 24-h incubation. The presented diagram is an average of five ELISA. FIGURE 5. Induction by IL-10 with or without LPS of CXCL13 in ELISA. A, Production of CXCL13 by monocyte-derived DC after 18-h CXCL13 measured by ELISA) (Fig. 7A). The dose-response rela- incubation. Upper panel, Seven donors in whom CXCL13 was not induced tionship of chemotaxis in response to supernatants form LPS/IL- by LPS alone; Lower panel, Three donors who did respond to LPS alone. 10-stimulated DC was typically bell-shaped (not shown). The B B, Production of CXCL13 by pDC and myeloid DC. Results are an average of three independent donors. cell DHL-4 follicular lymphoma line was also tested. DHL-4 cells show a significant migration in response to supernatants containing CXCL13, as well as to recombinant CXCL13, and this migration was significantly blocked by a specific anti-CXCL13 mAb, but not per-repressed). Thus, transcriptional profiling reveals on a genome by an irrelevant mAb. No migration was observed with control wide scale the complexity of the action of IL-10 and of its inter- supernatants (Fig. 7B). These results confirmed that DC treated play with LPS in DC, which goes beyond its classic immunosup- with LPS plus IL-10 produce a functionally active CXCL13. pressive and anti-inflammatory activity. Examination of genes regulated by IL-10 alone or in concert with LPS revealed the Discussion activation of distinct functional programs related to: the inhibition The general objective of the present study was to conduct a ge- of inflammation and immunity and the regulation of tissue remod- nome wide analysis of the effects of IL-10 on DC and of the in- elling; the tuning of cytokine/growth factor receptors and GPCR; terplay between IL-10 and LPS. In agreement with previous stud- and the stimulation of B cell function and lymphoid tissue neo- ies (30–32), LPS regulated the expression of a substantial fraction genesis. The discussion that follows will develop along this con- of the transcriptome, while IL-10 modulated a limited number of ceptual framework. genes. As expected, when IL-10 and LPS were combined, inhibi- The LPS-induced maturation of DC is associated with the aug- tion of LPS-induced regulation (positive or negative) was observed mented expression of surface molecules (e.g., CD86 and CD83), for a subset of genes. Moreover, DC exposed to combined LPS and production of cytokines (e.g., IL-12) and chemokines, and up-reg- IL-10 showed a distinct transcriptional profile, with a set of genes ulation of the chemokine receptor CCR7. In agreement with pre- being uniquely modulated by the two signals (superinduced or su- vious reports (6, 12–17, 35), IL-10 per se did not induce these 7040 IL-10-MODULATED TRANSCRIPTION IN DC

FIGURE 7. Chemotactic activity of supernatants of DC cultured for 18 h with LPS and IL-10. A, Migration of freshly isolated tonsillary B lymphocytes. CTRL supernatants (diluted 1/3) are from unstimulated DC. Results shown are net numbers of migrated cells in 10 HPF; basal migration of B lymphocytes was 7 Ϯ 1.5 cells. B, Migration of DHL-4 B cell lymphoma toward supernatants of untreated DC (CTRL), or DC treated with LPS plus IL-10 and toward recombinant CXCL13 (10 ng/ml). Mi- gration was partially blocked by an anti-CXCL13 mAb. Results shown are net numbers of migrated cells in 10 HPF; basal migration of DHL-4 cells was 50 Ϯ 6 cells. Results are from two representative experiments performed with tonsillary B cells, and three representative experiments with DHL-4 cells. FIGURE 6. Role of IL-10 in CXCL13 induction by LPS in DC. A, Correlation between the production of CXCL13 and the production of IL-10 by monocyte-derived DC after treatment with LPS. Twelve different donors were tested. B, Inhibition by anti-IL-10 mAb of CXCL13 production induced by LPS. gram of polarized type II cells which are oriented to tissue remodeling. IL-10 regulated the expression of a set of genes involved in genes in DC and inhibited as expected the LPS-induced stimula- signal transduction. IL-10 alone, or in combination with LPS aug- tion. IL-10 induced transcript expression of the surface molecule mented expression of two tyrosin phosphatases, PTPN1 and SLAM/CD150 and superinduced it with LPS, as also recently re- PTPN2. Although little is known about PTPN2, PTPN1 is known ported (36). SLAM is a member of the CD2 subfamily of the Ig to attenuate the signaling by dephosphorylating tyrosine residues superfamily (37) and its expression on DC was shown to be asso- located in the tyrosine kinase domain of the receptor itself and by ciated with maturation (38, 39). By engaging in homotypic inter- reducing the extent of Janus kinase (JAK) 2 phosphorylation (46). actions, it activates T cells (40). The fact that it is induced by IL-10 IL-10 also augmented the expression of RGS16 and SOCS3, in DC raises the possibility that this molecule may also be impor- whereas it inhibited PI3K␥ and mitogen-activated protein kinase tant for the activation of TH2 cells or T regulatory cells. Further- kinase kinase 4. The latter gene was up-regulated by LPS, whereas more, SLAM, in its soluble and membrane-bound forms, stimu- RGS16 and SOCS3 were superinduced by combined IL-10 and lates B cell proliferation and Ig synthesis (41). LPS. Induction by IL-10 and superinduction by IL-10 and LPS was IL-10 is an immunosuppressive cytokine which inhibits the mat- confirmed for SOCS3 at the protein level, as previously described uration and function of DC. Interestingly, IL-10 augmented ex- in neutrophils and in mononuclear phagocytes (28, 47–49). RGS ␣ pression of the vitamin D3 receptor. Vitamin D3 has been shown to proteins inhibit signaling through GPCR by binding to G , stim- affect DC differentiation and function (42, 43). Therefore, by up- ulating GTP hydrolysis and thereby reverting G protein activation. regulating the vitamin D3 receptor, IL-10 may promote a comple- RGS16 has recently been shown to attenuate signaling by chemo- mentary pathway of DC inhibition. kine receptors (50). Like PTPN1, SOCS3 is an inhibitor of the A set of genes regulated by IL-10 alone, or in combination with JAK-STAT pathway. It inhibits the tyrosine kinase activity of LPS, are related to the extracellular matrix and its remodeling. JAK2 (51, 52). Chemokine receptors have been shown to activate IL-10 augmented the expression of versican, PTX3, and ␣1-anti- the JAK-STAT pathway, which has been suggested to play a key trypsin. PTX3 is a unique soluble PPR (44, 45) which plays a role in chemotactic responses (53, 54). PI3K␥ is a key nonredun- nonredundant role in the assembly of the extracellular matrix of dant element in the signaling cascade activated by chemokine re- the cumulus oophorus and hence, in female fertility (45). Recent ceptors in various cell types (55–57), including DC (S. Sozzani results show that PTX3 is a constituent of the extracellular matrix and A. Del Prete, unpublished data). Interestingly, DC exposed to (81). IL-10 is produced by polarized M2 or alternatively activated high levels of primary inflammatory signals, such as LPS and IL- macrophages. These cells are geared to tissue remodeling (18, 19). 10, retain high levels of inflammatory chemokine receptors but do Induction by IL-10 of components of the extracellular matrix and not migrate in response to appropriate agonists (16, 23). Down- proteolytic enzyme inhibitors may be part of the functional pro- regulation of PI3K␥ and up-regulation of RGS16 and SOCS3 may The Journal of Immunology 7041 attenuate chemokine receptor signaling in DC exposed to IL-10 9. Muzio, M., D. Bosisio, N. Polentarutti, G. D’Amico, A. Stoppacciaro, and LPS. R. Mancinelli, C. van’t Veer, G. Penton-Rol, L. P. Ruco, P. Allavena, and A. Mantovani. 2000. Differential expression and regulation of Toll-like receptors A set of IL-10-modulated genes in DC was related to B cell (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. development and function. B cells are a major target for the action J. Immunol. 164:5998. 10. Jarrossay, D., G. Napolitani, M. Colonna, F. Sallusto, and A. Lanzavecchia. 2001. of IL-10. IL-10 acts as a potent growth and differentiation factor Specialization and complementarity in microbial molecule recognition by human Ϫ/Ϫ for activated B cells (17, 58–60). In fact, IL-10 mice are char- myeloid and plasmacytoid dendritic cells. Eur. J. Immunol. 31:3388. acterized by a reduction of B cells (61). Together with CD40L and 11. Allavena, P., A. Sica, A. Vecchi, M. Locati, S. Sozzani, and A. Mantovani. 2000. The chemokine receptor switch paradigm and dendritic cell migration: its sig- IL-2, IL-10 stimulates the proliferation of naive B cells and their nificance in tumor tissues. Immunol. Rev. 177:141. differentiation to plasma cells and memory cells (62–64). The re- 12. Buelens, C., V. Verhasselt, D. De Groote, K. Thielemans, M. Goldman, and sults presented here show that IL-10 induces in DC the expression F. Willems. 1997. Interleukin-10 prevents the generation of dendritic cells from human peripheral blood mononuclear cells cultured with interleukin-4 and gran- of a set of genes related to B cell differentiation and function, ulocyte/macrophage-colony-stimulating factor. Eur. J. Immunol. 27:756. which include SLAM, IL-7, pre-B cell colony-enhancing factor, 13. Allavena, P., L. Piemonti, D. Longoni, S. Bernasconi, A. Stoppacciaro, L. Ruco, and the chemokine, CXCL13. and A. Mantovani. 1998. Interleukin-10 prevents the differentation of monocytes to dendritic cells but promotes their maturation to macrophages. Eur. J. Immunol. Unexpectedly, IL-10 in combination with TLR engagement in- 28:359. creased expression and release of the chemokine CXCL13 in 14. Jonuleit, H., E. Schmitt, K. Steinbrink, and A. H. Enk. 2001. Dendritic cells as a monocyte-derived DC and in pDC. IL-10 generally suppresses tool to induce anergic and regulatory T cells. Trends Immunol. 22:394. 15. Corinti, S., C. Albanesi, A. la Sala, S. Pastore, and G. Girolomoni. 2001. Reg- production of inflammatory chemokines in DC as well as in other ulatory activity of autocrine IL-10 on dendritic cell functions. J. Immunol. cell types (17, 19). Exceptions to this general rule are CCL18 (20), 166:4312. CCL16 (21) and, as demonstrated here, CXCL13. Moreover, the 16. D’Amico, G., G. Frascaroli, G. Bianchi, P. Transidico, A. Doni, A. Vecchi, S. Sozzani, P. Allavena, and A. Mantovani. 2000. Uncoupling of inflammatory correlation between CXCL13 and IL-10 production in response to chemokine receptors by IL-10: generation of functional decoys. Nat. Immunol. LPS as well as Ab blocking experiments suggest that LPS-induced 1:387. endogenous IL-10 is key to CXCL13 production by DC. 17. Moore, K. W., R. de Waal Malefyt, R. L. Coffman, and A. O’Garra. 2001. In- terleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19:683. CXCL13 is primarily produced by follicular-type DC, although 18. Gordon, S. 2003. Alternative activation of macrophages. Nat Rev Immunol. 3:23. ϩ ϩ also CD1 CD4 myeloid DC localized in the germinal centers, 19. Mantovani, A., S. Sozzani, M. Locati, P. Allavena, and A. Sica. 2002. Macro- and myeloid DC exposed to TNF-␣ and IL-1␤, produce it (65–68). phage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol. 23:549. CXCL13 interacts with the CXCR5 receptor, expressed on B cells 20. Vulcano, M., S. Struyf, P. Scapini, M. Cassatella, S. Bernasconi, R. Bonecchi, and on a subset of CD4ϩ T cells which home to B cell follicles (66, A. Calleri, G. Penna, L. Adorini, W. Luini, et al. 2003. Unique regulation of 67) and provide help for Ig production (69, 70). Therefore, CCL18 production by maturing dendritic cells. J. Immunol. 170:3843. ϩ 21. Hedrick, J. A., A. Helms, A. Vicari, and A. Zlotnik. 1998. Characterization of a CXCL13 facilitates a three party interaction among DC, CXCR5 novel CC chemokine, HCC-4, whose expression is increased by interleukin-10. follicular-type T helper cells, and B cells (71). Blood 91:4242. Mice lacking CXCR5 fail to develop B cell follicles in the 22. Sozzani, S., S. Ghezzi, G. Iannolo, W. Luini, A. Borsatti, N. Polentarutti, A. Sica, M. Locati, C. Mackay, T. N. C. Wells, et al. 1998. Interleukin-10 increases CCR5 spleen and in Peyer’s patches and CXCR5 and its cognate ligand, expression and HIV infection in human monocytes. J. Exp. Med. 187:439. CXCL13, are part of a cascade which includes IL-7 and lympho- 23. Mantovani, A., M. Locati, A. Vecchi, S. Sozzani, and P. Allavena. 2001. Decoy receptors: a strategy to regulate inflammatory cytokines and chemokines. Trends toxin, leading to the organization of secondary lymphoid tissue Immunol. 22:328. (72–75). In addition to CXCL13, IL-10 induces in DC the expres- 24. Piemonti, L., S. Bernasconi, W. Luini, Z. Trobonjaca, A. Minty, P. Allavena, and sion of IL-7, a cytokine first identified based on its capacity to A. Mantovani. 1995. IL-13 supports differentiation of dendritic cells from circulating precursors in concert with GM-CSF. Eur. Cytokine Network 6:245. induce the growth of immature B lymphocytes (76). Hence IL-10, 25. D’Amico, G., G. Bianchi, S. Bernasconi, L. Bersani, L. Piemonti, S. Sozzani, alone or in costimulation with TLR-ligands, induces in DC two A. Mantovani, and P. Allavena. 1998. Adhesion, transendothelial migration, and mediators, IL-7 and CXCL13, which are key to the organization of reverse transmigration of in vitro cultured dendritic cells. Blood 92:207. 26. Locati, M., U. Deuschle, M. L. Massardi, F. O. Martinez, M. Sironi, S. Sozzani, secondary lymphoid tissues. CXCL13 is also expressed in ectopic T. Bartfai, and A. Mantovani. 2002. Analysis of the gene expression profile lymphoid tissue under chronic inflammatory conditions, for exam- activated by the CC chemokine ligand 5/RANTES and by lipopolysaccharide in ple, within the synovium of chronic arthritis patients (77), in human monocytes. J. Immunol. 168:3557. 27. Wodicka, L., H. Dong, M. Mittmann, M. H. Ho, and D. J. Lockhart. 1997. Sjo¨gren syndrome (78), and in aberrant gut-associated lymphoid Genome-wide expression monitoring in Saccharomyces cerevisiae. Nat. Biotech- tissue (79, 80). Our findings that IL-10 amplifies the release of nol. 15:1359. functionally active CXCL13 by DC suggest that IL-10 produced in 28. Crepaldi, L., S. Gasperini, J. A. Lapinet, F. Calzetti, C. Pinardi, Y. Liu, S. Zurawski, R. de Waal Malefyt, K. W. Moore, and M. A. Cassatella. 2001. chronic inflammatory conditions may promote the organization of Up-regulation of IL-10R1 expression is required to render human neutrophils extranodal lymphoid follicles through increased release of fully responsive to IL-10. J. Immunol. 167:2312. CXCL13. 29. Peri, G., M. Introna, D. Corradi, G. Iacuitti, S. Signorini, F. Avanzini, F. Pizzetti, A. P. Maggioni, T. Moccetti, M. Metra, et al. 2000. PTX3, a prototypic long pentraxin, is an early indicator of acute myocardial infarction in man. Circulation References 102:636. 1. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of 30. Hashimoto, S. I., T. Suzuki, S. Nagai, T. Yamashita, N. Toyoda, and immunity. Nature 392:245. K. Matsushima. 2000. Identification of genes specifically expressed in human 2. Lanzavecchia, A., and F. Sallusto. 2001. The instructive role of dendritic cells on activated and mature dendritic cells through serial analysis of gene expression. T cell responses: lineages, plasticity, and kinetics. Curr. Opin. Immunol. 13:291. Blood 96:2206. 3. Steinman, R. M., D. Hawiger, K. Liu, L. Bonifaz, D. Bonnyay, K. Mahnke, 31. Granucci, F., C. Vizzardelli, E. Virzi, M. Rescigno, and P. Ricciardi-Castagnoli. T. Iyoda, J. Ravetch, M. Dhodapkar, K. Inaba, and M. Nussenzweig. 2003. Den- 2001. Transcriptional reprogramming of dendritic cells by differentiation stimuli. dritic cell function in vivo during the steady state: a role in peripheral tolerance. Eur. J. Immunol. 31:2539. Ann. NY Acad. Sci. 987:15. 32. Huang, Q., D. Liu, P. Majewski, L. C. Schulte, J. M. Korn, R. A. Young, 4. Shortman, K., and Y. J. Liu. 2002. Mouse and human dendritic cell subtypes. Nat. E. S. Lander, and N. Hacohen. 2001. The plasticity of dendritic cell responses to Rev. Immunol. 2:151. pathogens and their components. Science 294:870. 5. Schuler, G., B. Schuler-Thurner, and R. M. Steinman. 2003. The use of dendritic 33. Samal, B., Y. Sun, G. Stearns, C. Xie, S. Suggs, and I. McNiece. 1994. Cloning cells in cancer immunotherapy. Curr. Opin. Immunol. 15:138. and characterization of the cDNA encoding a novel human pre-B-cell colony- 6. Banchereau, J., S. Paczesny, P. Blanco, L. Bennett, V. Pascual, J. Fay, and enhancing factor. Mol. Cell. Biol. 14:1431. A. K. Palucka. 2003. Dendritic cells: controllers of the immune system and a new 34. Lanzavecchia, A., and F. Sallusto. 2001. Regulation of T cell immunity by den- promise for immunotherapy. Ann. NY Acad. Sci. 987:180. dritic cells. Cell 106:263. 7. Akira, S., and H. Hemmi. 2003. Recognition of pathogen-associated molecular 35. Koch, F., U. Stanzl, P. Jennewein, K. Janke, C. Heufler, E. Kampgen, N. Romani, patterns by TLR family. Immunol. Lett. 85:85. and G. Schuler. 1996. High level IL-12 production by murine dendritic cells: 8. Rescigno, M., F. Granucci, and P. Ricciardi-Castagnoli. 2000. Molecular events upregulation via MHC class II and CD40 molecules and downregulation by IL-4 of bacterial-induced maturation of dendritic cells. J Clin. Immunol. 20:161. and IL-10. J. Exp. Med. 184:741. 7042 IL-10-MODULATED TRANSCRIPTION IN DC

36. McBride, J. M., T. Jung, J. E. de Vries, and G. Aversa. 2002. IL-10 alters DC 59. Defrance, T., B. Vanbervliet, F. Briere, I. Durand, F. Rousset, and J. Banchereau. function via modulation of cell surface molecules resulting in impaired T-cell 1992. Interleukin 10 and transforming growth factor ␤ cooperate to induce anti- responses. Cell. Immunol. 215:162. CD40-activated naive human B cells to secrete immunoglobulin A. J. Exp. Med. 37. Veillette, A., and S. Latour. 2003. The SLAM family of immune-cell receptors. 175:671. Curr. Opin. Immunol. 15:277. 60. Briere, F., C. Servet-Delprat, J. M. Bridon, J. M. Saint-Remy, and J. Banchereau. ϩ ϩ 38. Kruse, M., E. Meinl, G. Henning, C. Kuhnt, S. Berchtold, T. Berger, G. Schuler, 1994. Human interleukin 10 induces naive surface immunoglobulin D (sIgD ) and A. Steinkasserer. 2001. Signaling lymphocytic activation molecule is ex- B cells to secrete IgG1 and IgG3. J. Exp. Med. 179:757. pressed on mature CD83ϩ dendritic cells and is up-regulated by IL-1␤. J. Im- 61. Poussin, M. A., E. Goluszko, T. K. Hughes, S. I. Duchicella, and P. Christadoss. munol. 167:1989. 2000. Suppression of experimental autoimmune myasthenia gravis in IL-10 gene- 39. Bleharski, J. R., K. R. Niazi, P. A. Sieling, G. Cheng, and R. L. Modlin. 2001. disrupted mice is associated with reduced B cells and serum cytotoxicity on Signaling lymphocytic activation molecule is expressed on CD40 ligand-acti- mouse cell line expressing AChR. J. Neuroimmunol. 111:152. vated dendritic cells and directly augments production of inflammatory cytokines. 62. Hauser, A. E., G. Muehlinghaus, R. A. Manz, G. Cassese, S. Arce, G. F. Debes, J. Immunol. 167:3174. A. Hamann, C. Berek, S. Lindenau, T. Doerner, et al. 2003. Long-lived plasma 40. Sidorenko, S. P., and E. A. Clark. 2003. The dual-function CD150 receptor sub- cells in immunity and inflammation. Ann. NY Acad. Sci. 987:266. family: the viral attraction. Nat. Immunol. 4:19. 63. Manz, R. A., S. Arce, G. Cassese, A. E. Hauser, F. Hiepe, and A. Radbruch. 2002. 41. Punnonen, J., B. G. Cocks, J. M. Carballido, B. Bennett, D. Peterson, G. Aversa, Humoral immunity and long-lived plasma cells. Curr. Opin. Immunol. 14:517. and J. E. de Vries. 1997. Soluble and membrane-bound forms of signaling lym- 64. Roy, M. P., C. H. Kim, and E. C. Butcher. 2002. Cytokine control of memory B phocytic activation molecule (SLAM) induce proliferation and Ig synthesis by cell homing machinery. J. Immunol. 169:1676. activated human B lymphocytes. J. Exp. Med. 185:993. 65. Vissers, J. L., F. C. Hartgers, E. Lindhout, C. G. Figdor, and G. J. Adema. 2001. 42. Piemonti, L., P. Monti, M. Sironi, P. Fraticelli, B. E. Leone, E. Dal Cin, BLC (CXCL13) is expressed by different dendritic cell subsets in vitro and in vivo. Eur. J. Immunol. 31:1544. P. Allavena, and V. Di Carlo. 2000. Vitamin D3 affects differentiation, maturation, and function of human monocyte-derived dendritic cells. J. Immunol. 66. Gunn, M. D., V. N. Ngo, K. M. Ansel, E. H. Ekland, J. G. Cyster, and 164:4443. L. T. Williams. 1998. A B-cell-homing chemokine made in lymphoid follicles ␣ activates Burkitt’s lymphoma receptor-1. Nature 391:799. 43. Penna, G., and L. Adorini. 2000. 1 ,25-Dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired 67. Legler, D. F., M. Loetscher, R. S. Roos, I. Clark-Lewis, M. Baggiolini, and alloreactive T cell activation. J. Immunol. 164:2405. B. Moser. 1998. B cell-attracting chemokine 1, a human CXC chemokine ex- 44. Mantovani, A., C. Garlanda, and B. Bottazzi. 2003. Pentraxin 3, a non-redundant pressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/ soluble pattern recognition receptor involved in innate immunity. Vaccine CXCR5. J. Exp. Med. 187:655. 21(Suppl. 2):543. 68. Ishikawa, S., S. Nagai, T. Sato, K. Akadegawa, H. Yoneyama, Y. Y. Zhang, N. Onai, and K. Matsushima. 2002. Increased circulating CD11bϩCD11cϩ den- 45. Garlanda, C., E. Hirsch, S. Bozza, A. Salustri, M. De Acetis, R. Nota, ␣ A. Maccagno, F. Riva, B. Bottazzi, G. Peri, et al. 2002. Non-redundant role of the dritic cells (DC) in aged BWF1 mice which can be matured by TNF- into long pentraxin PTX3 in anti-fungal innate immune response. Nature 420:182. BLC/CXCL13-producing DC. Eur. J. Immunol. 32:1881. 69. Breitfeld, D., L. Ohl, E. Kremmer, J. Ellwart, F. Sallusto, M. Lipp, and R. Forster. 46. Gu, F., N. Dube, J. W. Kim, A. Cheng, J. Ibarra-Sanchez Mde, M. L. Tremblay, 2000. Follicular B helper T cells express CXC chemokine receptor 5, localize to and Y. R. Boisclair. 2003. Protein tyrosine phosphatase 1B attenuates growth B cell follicles, and support immunoglobulin production. J. Exp. Med. 192:1545. hormone-mediated JAK2-STAT signaling. Mol. Cell. Biol. 23:3753. 70. Kim, C. H., L. S. Rott, I. Clark-Lewis, D. J. Campbell, L. Wu, and E. C. Butcher. 47. Berlato, C., M. A. Cassatella, I. Kinjyo, L. Gatto, A. Yoshimura, and F. Bazzoni. ϩ 2001. Subspecialization of CXCR5 T cells: B helper activity is focused in a 2002. Involvement of suppressor of cytokine signaling-3 as a mediator of the ϩ germinal center-localized subset of CXCR5 T cells. J. Exp. Med. 193:1373. inhibitory effects of IL-10 on lipopolysaccharide-induced macrophage activation. 71. Reif, K., E. H. Ekland, L. Ohl, H. Nakano, M. Lipp, R. Forster, and J. G. Cyster. J. Immunol. 168:6404. 2002. Balanced responsiveness to chemoattractants from adjacent zones deter- 48. Ito, S., P. Ansari, M. Sakatsume, H. Dickensheets, N. Vazquez, R. P. Donnelly, mines B-cell position. Nature 416:94. A. C. Larner, and D. S. Finbloom. 1999. Interleukin-10 inhibits expression of 72. Ohl, L., G. Henning, S. Krautwald, M. Lipp, S. Hardtke, G. Bernhardt, O. Pabst, both interferon-␣- and interferon-␥- induced genes by suppressing tyrosine phos- and R. Forster. 2003. Cooperating mechanisms of CXCR5 and CCR7 in devel- phorylation of STAT1. Blood 93:1456. opment and organization of secondary lymphoid organs. J. Exp. Med. 197:1199. 49. Petit-Bertron, A. F., C. Fitting, J. M. Cavaillon, and M. Adib-Conquy. 2003. 73. Luther, S. A., K. M. Ansel, and J. G. Cyster. 2003. Overlapping roles of Adherence influences monocyte responsiveness to interleukin-10. J. Leukocyte CXCL13, interleukin 7 receptor ␣, and CCR7 ligands in lymph node develop- Biol. 73:145. ment. J. Exp. Med. 197:1191. 50. Lippert, E., D. L. Yowe, J. A. Gonzalo, J. P. Justice, J. M. Webster, E. R. Fedyk, 74. Yoshida, H., A. Naito, J. Inoue, M. Satoh, S. M. Santee-Cooper, C. F. Ware, M. Hodge, C. Miller, J. C. Gutierrez-Ramos, F. Borrego, et al. 2003. Role of A. Togawa, and S. Nishikawa. 2002. Different cytokines induce surface lympho- regulator of G protein signaling 16 in inflammation-induced T lymphocyte mi- toxin-␣␤ on IL-7 receptor-␣ cells that differentially engender lymph nodes and gration and activation. J. Immunol. 171:1542. Peyer’s patches. Immunity 17:823. 51. Alexander, W. S. 2002. Suppressors of cytokine signalling (SOCS) in the im- 75. Drayton, D. L., X. Ying, J. Lee, W. Lesslauer, and N. H. Ruddle. 2003. Ectopic mune system. Nat. Rev. Immunol. 2:410. LT ␣␤ directs lymphoid organ neogenesis with concomitant expression of pe- 52. Chen, X. P., J. A. Losman, and P. Rothman. 2000. SOCS proteins, regulators of ripheral node addressin and a HEV-restricted sulfotransferase. J. Exp. Med. intracellular signaling. Immunity 13:287. 197:1153. 53. Soriano, S. F., P. Hernanz-Falcon, J. M. Rodriguez-Frade, A. M. De Ana, 76. Fleming, H. E., and C. J. Paige. 2002. Cooperation between IL-7 and the pre-B R. Garzon, C. Carvalho-Pinto, A. J. Vila-Coro, A. Zaballos, D. Balomenos, cell receptor: a key to B cell selection. Semin. Immunol. 14:423. A. C. Martinez, and M. Mellado. 2002. Functional inactivation of CXC chemo- 77. Shi, K., K. Hayashida, M. Kaneko, J. Hashimoto, T. Tomita, P. E. Lipsky, kine receptor 4-mediated responses through SOCS3 up-regulation. J. Exp. Med. H. Yoshikawa, and T. Ochi. 2001. Lymphoid chemokine B cell-attracting che- 196:311. mokine-1 (CXCL13) is expressed in germinal center of ectopic lymphoid follicles 54. Mellado, M., J. M. Rodriguez-Frade, S. Manes, and A. C. Martinez. 2001. Che- within the synovium of chronic arthritis patients. J. Immunol. 166:650. mokine signaling and functional responses: the role of receptor dimerization and 78. Amft, N., S. J. Curnow, D. Scheel-Toellner, A. Devadas, J. Oates, J. Crocker, TK pathway activation. Annu. Rev. Immunol. 19:397. J. Hamburger, J. Ainsworth, J. Mathews, M. Salmon, et al. 2001. Ectopic ex- 55. Sasaki, T., J. Irie-Sasaki, R. G. Jones, A. J. Oliveira-dos-Santos, W. L. Stanford, pression of the B cell-attracting chemokine BCA-1 (CXCL13) on endothelial B. Bolon, A. Wakeham, A. Itie, D. Bouchard, I. Kozieradzki, et al. 2000. Func- cells and within lymphoid follicles contributes to the establishment of germinal tion of PI3K␥ in thymocyte development, T cell activation, and neutrophil mi- center-like structures in Sjogren’s syndrome. Arthritis Rheum. 44:2633. gration. Science 287:1040. 79. Mazzucchelli, L., A. Blaser, A. Kappeler, P. Scharli, J. A. Laissue, M. Baggiolini, 56. Li, Z., H. Jiang, W. Xie, Z. Zhang, A. V. Smrcka, and D. Wu. 2000. Roles of and M. Uguccioni. 1999. BCA-1 is highly expressed in Helicobacter pylori- PLC-␤2 and -␤3 and PI3K␥ in chemoattractant-mediated signal transduction. induced mucosa-associated lymphoid tissue and gastric lymphoma. J. Clin. In- Science 287:1046. vest. 104:R49. 57. Hirsch, E., V. L. Katanaev, C. Garlanda, O. Azzolino, L. Pirola, L. Silengo, 80. Carlsen, H. S., E. S. Baekkevold, F. E. Johansen, G. Haraldsen, and S. Sozzani, A. Mantovani, F. Altruda, and M. P. Wymann. 2000. Central role for P. Brandtzaeg. 2002. B cell attracting chemokine 1 (CXCL13) and its receptor G protein-coupled phosphoinositide 3-kinase ␥ in inflammation. Science CXCR5 are expressed in normal and aberrant gut-associated lymphoid tissue. Gut 287:1049. 51:364. 58. Rousset, F., E. Garcia, T. Defrance, C. Peronne, N. Vezzio, D. H. Hsu, 81. Salustri, A., C. Garlanda, E. Hirsch, M. De Acetis, A. Maccagno, B. Bottazzi, R. Kastelein, K. W. Moore, and J. Banchereau. 1992. Interleukin 10 is a potent A. Doni, G. Mantovani, P. B. Peccoz, G. Salvatori, et al. 2004. PTX3 plays a key growth and differentiation factor for activated human B lymphocytes. Proc. Natl. role in the organization of the cumulus oophorus extracellular matrix and in in Acad. Sci. USA 89:1890. vivo fertilization. Development 131:1577