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The Journal of Immunology, 2004, 172: 7031-7042.
Copyright © 2004 by The American Association of Immunologists

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{dagger}, Massimo Locati{dagger}, Giancarlo Bianchi*, Manuela Nebuloni{ddagger}, Gianluca Vago{ddagger}, Flavia Bazzoni§, Silvano Sozzani*,¶, Paola Allavena* and Alberto Mantovani2,*,{dagger}

* Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; {dagger} Centro di Eccellenza per l’Innovazione Diagnostica e Terapeutica, Institute of General Pathology, University of Milan, Milan, Italy; {ddagger} Institute of Pathology, University of Milan, Ospedale Luigi Sacco, Milan, Italy; § Department of Pathology, University of Verona, Verona, Italy; and Department of Biotechnology and Biomedical Sciences, Section of General Pathology and Immunology, University of Brescia, Brescia, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To understand the modulation of dendritic cell (DC) function by IL-10, gene expression profiling was performed by using Affymetrix 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 {gamma} 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Dendritic cells (DC)3 are powerful APCs specialized in the priming of resting T cells and in the initiation of the immune response (1, 2). DC originate from bone marrow and circulating precursors and can be distinguished in at least two subsets: myeloid-derived DC and DC derived from nonmyeloid precursors, of which plasmacytoid DC (pDC) and Langerhans cells are the major components (2, 3, 4, 5, 6).

At their immature stage, DC are sparse in peripheral tissues and stand guard for the encounter of an Ag. DC express a rich repertoire of surface receptors such as Toll-like (TLR) and pattern recognition receptors (PRR) which allow the recognition of pathogen molecules (7, 8, 9, 10). The engagement of TLR and PRR on DC initiates a cascade of signaling events leading to the secretion of inflammatory and immuno-modulatory factors mediating protective immunity (7). For instance, activation of DC through engagement of TLR4 by LPS induces up-regulation of costimulatory and MHC molecules, of maturation markers such as CD83, DC-lysosome-associated membrane protein, and CCR7, increased secretion of cytokines and chemokines, and potent APC function (1, 2, 11).

The transition of resting to mature DC is a critical step for the optimal initiation of immunity. Several studies have provided evidence that activation/maturation of DC is a highly modulable process that can be accelerated or inhibited by several biological factors. One such critical factor is IL-10. IL-10 inhibits the differentiation of new DC from monocytic precursors and blocks DC maturation induced by LPS, as well as functional activities associated with their mature state (6, 12, 13, 14, 15, 16, 17).

IL-10 is a multifunctional cytokine whose general effects are aimed to limit the inflammatory response and prevent tissue damage. This is achieved by down-regulating the expression of inflammatory cytokines/chemokines, and inhibiting effector functions of T cells and mononuclear phagocytes (17). In contrast, IL-10, by suppressing Th1-polarized T cell responses and by inducing the differentiation of suppressor/T regulatory cells, can also inhibit the development of protective immunity. Thus, this cytokine plays a critical role in the balance between immune protection and pathology.

However, the effect of IL-10 in mononuclear phagocytes is not purely inhibitory. In fact, IL-10 increases the differentiation of monocytes into macrophages and enhances the endocytic ability of DC (13, 18, 19) and induces the production of selected chemokines such as CC chemokine ligand (CCL) 18 (20) and CCL16 (21). IL-10 also up-regulates expression of selected chemokine receptors (22). Moreover, DC exposed to a combination of IL-10 and the prototypic inflammatory signal, LPS, retain at their surface high levels of inflammatory chemokine receptors, which do not elicit migration and act as decoys and scavengers for inflammatory chemokines (16, 23).

In an effort to explore the complexity of the biological role of IL-10 on DC, we conducted a genome-wide analysis of gene expression by human myeloid DC upon exposure to IL-10. Furthermore, as during an inflammatory process, IL-10 is generated, and therefore, both proinflammatory and anti-inflammatory signals are locally present, we analyzed also the transcriptome of DC concomitantly treated with LPS and IL-10. Under these conditions, it is well known that several genes up-regulated by LPS are counterregulated by IL-10. In this paper, we focused our attention on those genes whose induction with LPS plus IL-10 treatment significantly differed from treatments with LPS or IL-10 alone.

In the complex interplay mediated by IL-10 with or without LPS, we identified at least four distinct functional programs: 1) genes related to the control of specific immunity and inflammation, 2) genes involved in tissue remodeling, 3) genes mediating the tuning of cytokine receptor and G protein-coupled receptor (GPCR) signaling, and 4) genes promoting B cell development/function and lymphoid tissue neogenesis. Finally, among genes related to B cell biology, we further studied the expression of the B cell attracting chemokine, CXC chemokine ligand (CXCL) 13, and demonstrated that IL-10 strongly augments the LPS-induced production of functionally active CXCL13 by DC. This novel finding reveals that IL-10 sustains humoral immunity by inducing the production, in APCs, of a chemokine which amplifies B cell recruitment and promotes lymphoid tissue neogenesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Preparation and treatment of DC

Cells were obtained as described (24, 25). Briefly, 95% pure monocytes (as assessed by CD14 analysis by flow cytometry) were obtained by using CD14 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). DC were obtained by culturing monocytes for 6 days at 1 x 106 cells/ml in RPMI 1640 (Biochrom, Berlin, Germany) with 10% FBS (HyClone, Logan, UT) containing 50 ng/ml GM-CSF (Novartis Pharmaceuticals, Basel, Switzerland) and 10 ng/ml IL-13 (a gift from Dr. A. Minty, Sanofi Elf Bio Recherches, Labège, France). Myeloid DC and pDC were obtained from buffy coats of healthy donors using the CD1c (BDCA-1) dendritic cell isolation and the BDCA-4 cell isolation kits (Miltenyi Biotec). LPS (Escherichia coli 055:B5; Sigma-Aldrich, St. Louis, MO) was used at 10 ng/ml; IL-10 (Schering-Plough, Kenilworth, NJ) was used at 50 ng/ml; and CpG oligonucleotides (ODN) motif GTCGTT (2006) was provided by Invitrogen Life Technologies (Rockville, MD) and used at 2 µg/ml.

cRNA preparation, hybridization, and microarrays analysis

For transcriptional profile analysis, DC of six healthy donors were prepared, resuspended at 1 x 106 cells/ml in RPMI 1640 supplemented with 10% FBS, and stimulated with IL-10, LPS, or a combination of both stimuli for 2 and 8 h. As quality control of the stimulation, phenotype analysis and functional activity of the cells were evaluated (16). Total RNA was obtained as described (26), quantified, and normalized among donors according to GAPDH levels, as determined by real-time PCR analysis. Two independent RNA pools of three donors each were labeled, processed, and independently hybridized on human genome U95Av2 arrays (HG-U95Av2; Affymetrix, Santa Clara, CA) containing 12,500 full-length genes, as previously described (26, 27). Scanned images were processed using Microarray Suite 5.0 (Affymetrix), and raw data were analyzed using Data Mining Tool 3.0 (Affymetrix). The transcriptional profiles obtained in this first set of DC preparations were confirmed in a subsequent set of three donors. Modulated genes were defined according to the following criteria: detection of p <= 0.005; 100 <= expression signal <= 9000; change confidence interval >99%; fold change in expression >2 and difference of expression >150 intensity arbitrary units. Only those genes whose modulation was consistently observed in each of the three donors pools were considered in our analysis and consequently presented in this work (Figs. 1 and 2, Tables II and III).



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  		FIGURE 1. General description of the expression profiles obtained after treatment of DC with LPS, IL-10, and a combination of the two stimuli. A, Number of genes which are increased (open bars) or decreased (filled bars) after 2 and 8 h. B, Overlap between the set of genes regulated by LPS, IL-10, and the combination of the two. Square areas are proportional to the number of modulated genes. Only the 8-h time point is shown, but similar data were obtained at 2 h.

 


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  		FIGURE 2. Interplay between LPS and IL-10 in the regulation of gene expression. Upper panel, Number of genes whose expression is increased or decreased by LPS in DC, and whose modulation is counterregulated under treatment by LPS plus IL-10. Lower panel, Number of genes which are synergistically increased or synergistically decreased after treatment by LPS plus IL-10, compared with single treatments after 2 and 8 h. The window in the upper right corner indicates the expression behavior of genes under single treatments (C (changed), modulated; NC (nonchanged), nonmodulated).

 

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  		Table II. IL-10-regulated genesa

 

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  		Table III. Interplay between LPS and IL-10 in the regulation of gene expressiona

 
Among the genes modulated in DC after combined treatment with LPS plus IL-10, we were interested in the genes falling into the following categories: 1) synergistically increased compared with single treatments, 2) synergistically decreased compared with single treatments, 3) increased by LPS and counterregulated by the addition of IL-10, and 4) decreased by LPS and counterregulated by the addition of IL-10.

Genes which were synergistically increased after LPS plus IL-10 compared with LPS or IL-10 alone had to meet the following three criteria: 1) intensity signal of LPS plus IL-10 > 2 times average of the intensity signals of untreated, LPS alone, and IL-10 alone; 2) intensity signal (average centered) of LPS plus IL-10 > 2 times maximum intensity signal (average centered) among untreated, LPS alone, and IL-10 alone; and 3) intensity signal (average centered) of LPS plus IL-10 > 100 plus average of the intensity signals (average centered) of untreated, LPS alone, and IL-10 alone. Genes which were synergistically decreased after LPS plus IL-10 compared with LPS or IL-10 alone had to meet the following three criteria: 1) intensity signal of LPS plus IL-10 < 0.5 times average of the intensity signals of untreated, LPS alone, and IL-10 alone; 2) intensity signal (average centered) of LPS plus IL-10 < 2 times minimum intensity signal (average centered) among untreated, LPS alone, and IL-10 alone; and 3) intensity signal (average centered) of LPS plus IL-10 < (Average of the intensity signals (average centered) of untreated, LPS alone and IL-10 alone) – 100.

The average centered signal can be mathematically defined as: original signal of the considered treatment – average of the original signals of ("untreated", "LPS", "IL-10", and "LPS plus IL-10").

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 expression is increased by LPS): 1) intensity signal of LPS/Intensity signal of LPS plus IL-10 > and 2) Intensity signal of LPS – Intensity signal of LPS plus IL-10 > 100. The criteria used for those genes whose expression is decreased by LPS were: 1) intensity signal of LPS/intensity signal of LPS plus IL-10 < 0.5 and 2) intensity signal of LPS plus IL-10 – intensity signal of LPS > 100.

Real-time PCR and Northern blot analysis

DC of three healthy donors, other than those used for transcriptional profiling, were exposed to LPS and IL-10 and checked for chemokine receptor expression and migration (16) as a quality control. Total RNA was obtained as described (26). Starting with 1 µg of total RNA, the synthesis of cDNA was performed by using the TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, CA). PCR was done using SYBR Green PCR Core Reagents mix (Applied Biosystems) containing 1x SYBR Green PCR buffer; 3 mM MgCl2; 100 mM dATP, dCTP, and dGTP; 200 mM dUTP; 0.025 U/ml AmpliTaq Gold DNA polymerase; 0.01 U/ml AmpErase UNG. Gene-specific forward and reverse primers were designed using the Primer Express software (Applied Biosystems) and were provided by Invitrogen (Carlsbad, CA). They were used at 2 pmol/ml in the reaction. Sequences are presented in Table I.


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  		Table I. Primers used for real-time PCR validation of the transcriptional profiles

 
The reaction conditions were as follows: 2 min at 50°C (1 cycle), 10 min at 95°C (1 cycle), 15 s at 95°C, and 1 min at 60°C (40 cycles). Gene-specific PCR products were continuously measured by means of an ABI PRISM 5700 detection system (PerkinElmer/Cetus, Norwalk, CT). Samples were normalized using the housekeeping gene, GAPDH. Triplicates were performed for each experimental point. Results are shown as a fold modulation over control.

In one case (SOCS3), the microarray analysis was validated by Northern blot after 2- and 8-h exposure to LPS, IL-10, and LPS plus IL-10. Total RNA was extracted by the TRIzol method, blotted, and hybridized as described (22). A specific SOCS3 probe was obtained by using the hSOCS MultiProbe Template Set (BD PharMingen, San Diego, CA), following the instructions of the manufacturer, and labeled by Megaprime DNA labeling system (Amersham, Buckinghamshire, U.K.) with [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham). Membranes were prehybridized at 42°C in Hybrisol (Oncor, Gaithersburg, MD) and hybridized overnight with 1 x 106 cpm/ml 32P-labeled probe. Membranes were then washed 3 times with 2x SSC (1x SSC = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), at room temperature for 10 min, twice with 2x SSC, 1% SDS at 60°C for 20 min, and then with 0.1x SSC for 5 min, before being exposed to XAR-5 films (Kodak, Rochester, NY) using intensifier screens at –80°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, Birmingham, 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 (Immuno-Biological Laboratories, Tokyo, Japan) diluted at 5 µg/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 1x 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 x 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 µg/ml, mouse IgG2B, clone 23738; R&D Systems). An anti-CCR2 mAb (2 µg/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 using the B cell separation kit (Miltenyi Biotec), following the instructions of the manufacturer. Migration assays were conducted for 3 h using 48-well chambers (NeuroProbe, Cabin John, MD) with 8-µm polyvinylpyrrolidone-free filters coated with 10 µg/ml fibronectin, as previously described (16). B lymphocytes were preincubated in migration medium (RPMI 1640, supplemented with 2.5% FBS) for 2 h and seeded (1.5 x 105/well). Recombinant human CXCL13 (R&D Systems), CXCL12 (PeproTech, Rocky Hill, NJ) and DC supernatants were used as chemotactic factors. To inhibit the migration in response to CXCL13, a specific mAb (clone 53610.11, mouse IgG1; R&D Systems) was placed at 20 µg/ml in the lower compartment of the chemotactic chamber. An isotype-matched murine mAb was used as negative control. Results presented are numbers of cells counted in 10 high power fields (HPF). CXCL12 (300 ng/ml) or CXCL13 (10 ng/ml) were used as positive controls.

Statistical analysis

Statistical analysis of the microarray experiment has been extensively explained above. Data of real-time PCR, ELISA, and chemotaxis experiments are expressed as the mean values ± SE. Significance was assessed by using the Student’s t test. For correlation analysis presented in Fig. 7A, R2 was calculated as the correlation coefficient of the linear regression and p value was determined by a Student’s paired t test.



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  		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 ± 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). Migration 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 ± 6 cells. Results are from two representative experiments performed with tonsillary B cells, and three representative experiments with DHL-4 cells.

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distinct but overlapping transcriptional profiles of DC exposed to LPS and IL-10

Fig. 1 provides an overview of the transcriptional profile of DC exposed to LPS, IL-10 or a combination of the two. In agreement with previous reports (30, 31, 32), LPS had a profound effect on the transcriptome expressed in human DC. By the stringent criteria used in the present study, after 2 h of exposure, LPS reproducibly increased expression of 177 genes and decreased that of 216 genes. Comparable numbers of modulated genes were observed at the 8-h time point. In contrast, IL-10 regulated expression of a restricted set of genes, ~10-fold lower than that modulated by LPS. With the stringent criteria used in the present study, the number of genes augmented by IL-10 was 26 and 19 at 2 and 8 h, respectively, whereas the decreased ones were 9 and 12 at 2 and 8 h, respectively. The transcriptional profile of IL-10-treated DC was distinct from that activated by LPS, with limited overlap (Fig. 1B). For instance, at the 8-h time point, only 8 of the 19 IL-10-regulated genes were also induced by LPS. The overall transcriptional profile of cells exposed to combined LPS plus IL-10 partially overlapped with, yet was distinct from, that of cells treated with LPS alone. For instance, at 8 h, overall 474 genes were positively or negatively regulated by combined LPS and IL-10, compared with 531 for LPS and 31 for IL-10. For the 8-h time point, 76 genes induced by IL-10 plus LPS were not stimulated by LPS or IL-10 alone.

Transcripts regulated by IL-10 with or without LPS

Several studies have analyzed the transcriptional profile of LPS-stimulated monocytes or DC (26, 30, 31, 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-{gamma}, and IFN-{beta}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{alpha} subunit and pre-B colony enhancing factor. The latter is a cytokine enhancing the proliferative 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 {gamma} isoform (PI3K{gamma}) 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-{beta}), 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 expression of 38 and 32 genes at 2 and 8 h, respectively, and additive negative effects on two genes for each time point. In addition, 22 and 14 genes not significantly affected by the individual agents were induced by the combined exposure to IL-10 and LPS, after 2 and 8 h, respectively. The genes induced in an additive or synergistic way by combined treatment with LPS and IL-10 included the pre-B colony-enhancing factor, the B cell attractant 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{gamma} was modulated by IL-10, and the effect of IL-10 was not significantly modified by LPS (Table II). PTX3 was stimulated by IL-10 and by LPS, although the synergistic effect was not scored significant (Table II).

Validation of the microarray transcriptional profile

In an effort to validate the microarray transcriptional analysis, real-time PCR was performed on DC obtained from three individual donors other than those investigated in the gene chip studies. We selected six genes whose induction by LPS was inhibited by concomitant exposure to IL-10 (referred to as "counterregulated genes") and five superinduced by combined LPS and IL-10 (referred to as "superinduced genes"). As shown in Fig. 3A, by real-time PCR analysis expression of CCR7, IL-12B, IFN-{beta}1, SERPINB2, TUBB2, and NR4A3 was augmented by exposure to LPS and inhibited by concomitant IL-10. For instance, expression of CCR7 was increased to 260.7 ± 57.7 arbitrary units by LPS and suppressed to 54.1 ± 11.5 arbitrary units by the addition of IL-10. In contrast, caspase 5, CXCL13, RGS16, versican, and TNFRSF1B were induced by LPS and their expression was superinduced by exposure to LPS and IL-10. RGS16 and versican were also induced by IL-10 alone. Moreover we took advantage of availability of blotted RNA samples and a specific probe from prior independent experiments to validate the regulation of SOCS3 by Northern blot analysis. As shown in Fig. 3B, SOCS3 mRNA was induced by LPS and IL-10 and superinduced by a combination of the two. Therefore, real-time PCR and Northern blot analysis on a set of 12 genes studied in three independent donors validated the transcriptional profiling.



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  		FIGURE 3. Validation by real-time PCR and Northern blot analysis of transcriptional profiles. A, The modulation of the expression of SERPINB2, IFN-{beta}1 (IFN-B1), IL-12B (p40), {beta}-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 ± SE values of three independent experiments with healthy donors. *, Significantly different from LPS treatment (p < 0.05). #, Significantly different from LPS and IL-10 single treatments (p < 0.05). B, Northern blot analysis of SOCS3 expression, representative of three experiments.

 
Protein production: superinduction of CXCL13

A series of modulated genes, selected among the unexpected IL-10-inducible or -superinducible genes, were investigated at the protein level. As shown in Fig. 4, in agreement with microarray analysis, IL-10 alone or in concert with LPS induced protein expression of SOCS3 (Western blot). The up-regulation of PTX3 after IL-10 alone was also confirmed (ELISA). Increased expression of SLAM protein (flow cytometry) was observed only after the combined treatment with LPS and IL-10.



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  		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.

 
Microarray and real-time PCR analysis unexpectedly revealed that IL-10 superinduces CXCL13 in human monocyte-derived DC (see above). Because IL-10 positively influences only a restricted set of (for instance CCL18 (20) and CCL16 (21)) it was important to assess the actual significance of this observation. CXCL13 was measured by ELISA in a set of 12 donors. As shown in Fig. 5A, IL-10 alone never induced appreciable amounts of CXCL13 under these conditions. LPS induced production of this chemokine (>0.6 ng/ml) in 5 of 12 donors. In both LPS-responsive and -nonresponsive DC, IL-10 significantly induced, in concert with LPS, or amplified LPS-induced production of CXCL13. When freshly isolated monocytes were tested, LPS induced the release of a significant amount of CXCL13 (1.5 ± 0.2 ng/ml) in three independent experiments (not shown). Surprisingly, in this cellular context, IL-10 did not synergize with LPS in the production of CXCL13.



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  		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 incubation. Upper panel, Seven donors in whom CXCL13 was not induced by LPS alone; Lower panel, Three donors who did respond to LPS alone. B, Production of CXCL13 by pDC and myeloid DC. Results are an average of three independent donors.

 
DC are heterogeneous and pDC represent a phenotypically and functionally distinct subset (1, 4, 34). We therefore examined the effect of IL-10 on human pDC. pDC express high levels of TLR9 and low levels of TLR4. Therefore, a CpG oligonucleotide (ODN motif GTCGTT (2006)) was used as agonist in these experiments. In a series of three donors (Fig. 5B), the CpG ODN induced low levels (1.0 ± 0.4 ng/ml) of CXCL13 in pDC. IL-10, inactive per se, dramatically increased CXCL13 production in pDC (7.1 ± 1.8 ng/ml, p < 0.01). Freshly isolated blood myeloid DC behaved like monocyte-derived DC when challenged with LPS and IL-10 (Fig. 5B).

These results indicated that indeed IL-10 costimulates CXCL13 production in DC in concert with TLR engagement. As in some experiments, LPS alone induced a considerable release of CXCL13 (Fig. 5), we considered whether the LPS-induced endogenous IL-10 could play a role in CXCL13 production. Therefore, we measured IL-10 in the supernatants of DC from 12 donors who had been stimulated with LPS and evaluated whether there was a correlation between endogenous production of IL-10 and CXCL13. As shown in Fig. 6A, there was a significant (p < 0.05), though far from strict (R2 = 0.332) correlation between IL-10 levels and CXCL13 levels in this series of 12 donors. To directly assess the role of endogenous IL-10 in LPS induction of CXCL13, a blocking mAb was used. In a series of three independent experiments, anti-IL-10 significantly reduced the LPS induction of CXCL13 in DC, confirming the important costimulatory role of IL-10 (Fig. 6B).



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  		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.

 
To test the functional activity of CXCL13 produced by DC treated with LPS and IL-10, we performed chemotaxis assays using supernatants from DC stimulated with IL-10 and LPS. Freshly isolated tonsillary B lymphocytes significantly responded to CXCL13-containing supernatants but not to control supernatant. The highest migration was observed with 1/3 dilution ({approx}0.5 ng/ml CXCL13 measured by ELISA) (Fig. 7A). The dose-response relationship of chemotaxis in response to supernatants form LPS/IL-10-stimulated DC was typically bell-shaped (not shown). The B 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 by an irrelevant mAb. No migration was observed with control supernatants (Fig. 7B). These results confirmed that DC treated with LPS plus IL-10 produce a functionally active CXCL13.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The general objective of the present study was to conduct a genome wide analysis of the effects of IL-10 on DC and of the interplay between IL-10 and LPS. In agreement with previous studies (30, 31, 32), LPS regulated the expression of a substantial fraction of the transcriptome, while IL-10 modulated a limited number of genes. As expected, when IL-10 and LPS were combined, inhibition of LPS-induced regulation (positive or negative) was observed for a subset of genes. Moreover, DC exposed to combined LPS and IL-10 showed a distinct transcriptional profile, with a set of genes being uniquely modulated by the two signals (superinduced or super-repressed). Thus, transcriptional profiling reveals on a genome wide scale the complexity of the action of IL-10 and of its interplay with LPS in DC, which goes beyond its classic immunosuppressive and anti-inflammatory activity. Examination of genes regulated by IL-10 alone or in concert with LPS revealed the activation of distinct functional programs related to: the inhibition of inflammation and immunity and the regulation of tissue remodelling; the tuning of cytokine/growth factor receptors and GPCR; and the stimulation of B cell function and lymphoid tissue neogenesis. The discussion that follows will develop along this conceptual framework.

The LPS-induced maturation of DC is associated with the augmented expression of surface molecules (e.g., CD86 and CD83), production of cytokines (e.g., IL-12) and chemokines, and up-regulation of the chemokine receptor CCR7. In agreement with previous reports (6, 12, 13, 14, 15, 16, 17, 35), IL-10 per se did not induce these genes in DC and inhibited as expected the LPS-induced stimulation. IL-10 induced transcript expression of the surface molecule SLAM/CD150 and superinduced it with LPS, as also recently reported (36). SLAM is a member of the CD2 subfamily of the Ig superfamily (37) and its expression on DC was shown to be associated with maturation (38, 39). By engaging in homotypic interactions, it activates T cells (40). The fact that it is induced by IL-10 in DC raises the possibility that this molecule may also be important for the activation of TH2 cells or T regulatory cells. Furthermore, SLAM, in its soluble and membrane-bound forms, stimulates B cell proliferation and Ig synthesis (41).

IL-10 is an immunosuppressive cytokine which inhibits the maturation and function of DC. Interestingly, IL-10 augmented expression of the vitamin D3 receptor. Vitamin D3 has been shown to affect DC differentiation and function (42, 43). Therefore, by up-regulating the vitamin D3 receptor, IL-10 may promote a complementary pathway of DC inhibition.

A set of genes regulated by IL-10 alone, or in combination with LPS, are related to the extracellular matrix and its remodeling. IL-10 augmented the expression of versican, PTX3, and {alpha}1-antitrypsin. PTX3 is a unique soluble PPR (44, 45) which plays a nonredundant role in the assembly of the extracellular matrix of the cumulus oophorus and hence, in female fertility (45). Recent results show that PTX3 is a constituent of the extracellular matrix (81). IL-10 is produced by polarized M2 or alternatively activated macrophages. These cells are geared to tissue remodeling (18, 19). Induction by IL-10 of components of the extracellular matrix and proteolytic enzyme inhibitors may be part of the functional program of polarized type II cells which are oriented to tissue remodeling.

IL-10 regulated the expression of a set of genes involved in signal transduction. IL-10 alone, or in combination with LPS augmented expression of two tyrosin phosphatases, PTPN1 and PTPN2. Although little is known about PTPN2, PTPN1 is known to attenuate the signaling by dephosphorylating tyrosine residues located in the tyrosine kinase domain of the receptor itself and by reducing the extent of Janus kinase (JAK) 2 phosphorylation (46). IL-10 also augmented the expression of RGS16 and SOCS3, whereas it inhibited PI3K{gamma} and mitogen-activated protein kinase kinase kinase 4. The latter gene was up-regulated by LPS, whereas RGS16 and SOCS3 were superinduced by combined IL-10 and LPS. Induction by IL-10 and superinduction by IL-10 and LPS was confirmed for SOCS3 at the protein level, as previously described in neutrophils and in mononuclear phagocytes (28, 47, 48, 49). RGS proteins inhibit signaling through GPCR by binding to G{alpha}, stimulating GTP hydrolysis and thereby reverting G protein activation. RGS16 has recently been shown to attenuate signaling by chemokine receptors (50). Like PTPN1, SOCS3 is an inhibitor of the JAK-STAT pathway. It inhibits the tyrosine kinase activity of JAK2 (51, 52). Chemokine receptors have been shown to activate the JAK-STAT pathway, which has been suggested to play a key role in chemotactic responses (53, 54). PI3K{gamma} is a key nonredundant element in the signaling cascade activated by chemokine receptors in various cell types (55, 56, 57), including DC (S. Sozzani and A. Del Prete, unpublished data). Interestingly, DC exposed to high levels of primary inflammatory signals, such as LPS and IL-10, retain high levels of inflammatory chemokine receptors but do not migrate in response to appropriate agonists (16, 23). Down-regulation of PI3K{gamma} and up-regulation of RGS16 and SOCS3 may attenuate chemokine receptor signaling in DC exposed to IL-10 and LPS.

A set of IL-10-modulated genes in DC was related to B cell development and function. B cells are a major target for the action of IL-10. IL-10 acts as a potent growth and differentiation factor for activated B cells (17, 58, 59, 60). In fact, IL-10–/– mice are characterized by a reduction of B cells (61). Together with CD40L and IL-2, IL-10 stimulates the proliferation of naive B cells and their differentiation to plasma cells and memory cells (62, 63, 64). The results presented here show that IL-10 induces in DC the expression of a set of genes related to B cell differentiation and function, which include SLAM, IL-7, pre-B cell colony-enhancing factor, and the chemokine, CXCL13.

Unexpectedly, IL-10 in combination with TLR engagement increased expression and release of the chemokine CXCL13 in monocyte-derived DC and in pDC. IL-10 generally suppresses production of inflammatory chemokines in DC as well as in other cell types (17, 19). Exceptions to this general rule are CCL18 (20), CCL16 (21) and, as demonstrated here, CXCL13. Moreover, the correlation between CXCL13 and IL-10 production in response to LPS as well as Ab blocking experiments suggest that LPS-induced endogenous IL-10 is key to CXCL13 production by DC.

CXCL13 is primarily produced by follicular-type DC, although also CD1+ CD4+ myeloid DC localized in the germinal centers, and myeloid DC exposed to TNF-{alpha} and IL-1{beta}, produce it (65, 66, 67, 68). CXCL13 interacts with the CXCR5 receptor, expressed on B cells and on a subset of CD4+ T cells which home to B cell follicles (66, 67) and provide help for Ig production (69, 70). Therefore, CXCL13 facilitates a three party interaction among DC, CXCR5+ follicular-type T helper cells, and B cells (71).

Mice lacking CXCR5 fail to develop B cell follicles in the spleen and in Peyer’s patches and CXCR5 and its cognate ligand, CXCL13, are part of a cascade which includes IL-7 and lymphotoxin, leading to the organization of secondary lymphoid tissue (72, 73, 74, 75). In addition to CXCL13, IL-10 induces in DC the expression of IL-7, a cytokine first identified based on its capacity to induce the growth of immature B lymphocytes (76). Hence IL-10, alone or in costimulation with TLR-ligands, induces in DC two mediators, IL-7 and CXCL13, which are key to the organization of secondary lymphoid tissues. CXCL13 is also expressed in ectopic lymphoid tissue under chronic inflammatory conditions, for example, within the synovium of chronic arthritis patients (77), in Sjögren syndrome (78), and in aberrant gut-associated lymphoid tissue (79, 80). Our findings that IL-10 amplifies the release of functionally active CXCL13 by DC suggest that IL-10 produced in chronic inflammatory conditions may promote the organization of extranodal lymphoid follicles through increased release of CXCL13.


   Footnotes
 
1 This work was supported by grants from the European Commission and MIUR-FIRB Grant RBNE01Y3N3. P.P. was supported by Grant 3235-57619.99 of the Swiss National Science Foundation. Transcriptional profiling was supported by Associazione Italiana Ricerca sul Cancro. Back

2 Address correspondence and reprint requests to Dr. Alberto Mantovani, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, I-20157 Milan, Italy. E-mail address: mantovani{at}marionegri.it Back

3 Abbreviations used in this paper: DC, dendritic cell; TLR, Toll-like receptor; PRR, pattern recognition receptor; CXCL, CXC chemokine ligand; GPCR, G protein-coupled receptor; SOCS3, suppressor of cytokine signaling 3; HPF, high power field; PTX3, pentraxin 3; CCL, CC chemokine ligand; PI3K{gamma}, phosphatidylinositol 3-kinase {gamma}; PTPN, protein-tyrosine phosphatase; SLAM, signaling lymphocytic-activating molecule; RGS16, regulator of G protein signaling 16; pDC, plasmacytoid DC; JAK, Janus kinase; ODN, oligonucleotide. Back

Received for publication December 12, 2003. Accepted for publication March 12, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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