Kinetics of IL-10-induced gene expression in human macrophages

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Immunobiology 210 (2005) 87–95 www.elsevier.de/imbio

Kinetics of IL-10-induced gene expression in human macrophages Taras T. Antoniva, Kyung-Hyun Park-Minb, Lionel B. Ivashkiva,b, a

Arthritis and Tissue Degeneration Program, Department of Medicine, Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021, USA b Graduate Program in Immunology, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA Received 16 March 2005; accepted 19 May 2005

Abstract IL-10 is a strong inhibitor of macrophage (MF) activation and inflammatory cytokine and chemokine production. To gain insight into mechanisms by which IL-10 suppresses inflammation, we performed a kinetic analysis of IL-10induced gene expression in primary human MF. IL-10 induced rapid and transient expression of genes encoding transcription factors, followed by sustained elevation or suppression of gene expression. IL-10 suppressed basal expression of interferon-inducible genes, suggesting that IL-10 interrupts autocrine interferon-mediated priming. IL-10 induced the expression of prostaglandin dehydrogenase (PGDH), the major catabolic enzyme involved in prostaglandin degradation. Concomitant with PGDH expression, IL-10 induced increased degradation of the inflammatory PGE2 and suppressed PGE2-mediated effects on MF morphology and gene expression. These results identify catabolism of inflammatory PGs as a mechanism of IL-10 anti-inflammatory action. r 2005 Elsevier GmbH. All rights reserved. Keywords: Expression profiling; Interleukin-10; Macrophages; Prostaglandin dehydrogenase

Introduction IL-10 is a potent anti-inflammatory cytokine and deactivator of macrophages (MF) and dendritic cells (Moore et al., 2001). Major suppressive functions of IL-10 include down-regulation of inflammatory cytokine and chemokine production, of production of ROIs, and Abbreviations: IGF1, insulin-like growth factor 1; MF, macrophage; PBMC, peripheral blood mononuclear cells; PGDH, 15-hydroxy prostaglandin dehydrogenase; PGE, E series prostaglandins Corresponding author. Arthritis and Tissue Degeneration Program, Department of Medicine, Hospital for Special Surgery, 535 East 70th Street, New York, NY 10021, USA. Tel.: +1 212 606 1653; fax: +1 212 774 2337. E-mail address: [email protected] (L.B. Ivashkiv). 0171-2985/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2005.05.003

of MHC expression. It has become clear that all IL-10 anti-inflammatory actions studied to date depend upon activation of the transcription factor Stat3, which is required for transcriptional responses to IL-10 (Riley et al., 1999; Takeda et al., 1999; Williams et al., 2004). Despite extensive efforts, the Stat3 target genes that mediate the anti-inflammatory effects of IL-10 have not been fully elucidated, although roles for Bcl3 and heme oxygenase 1 have been proposed (Kuwata et al., 2003; Lee and Chau, 2002). Gene expression profiling of IL-10 responses in human and murine MF has increased our understanding of IL-10 function, but it is not clear which of the many genes induced by IL-10 play the most important roles in mediating its anti-inflammatory effects (Jung et al., 2004; Lang et al., 2002; Williams et al., 2002).

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E series prostaglandins (PGEs) are potent inflammatory mediators whose synthesis is induced by many inflammatory factors such as IL-1 (Harris et al., 2002). PGEs are highly expressed at inflammatory sites in diseases like rheumatoid arthritis, and inhibitors of PG synthesis are effective in suppressing inflammation in these diseases (Turini and DuBois, 2002). PGEs are small lipid molecules derived by an enzymatic cascade that begins with phospholipase A-mediated release of arachidonic acid from membranes that is followed by modification by enzymes that include PGE synthetases and cyclooxygenases (Funk, 2001). Therapeutic targeting of PGs has been focused on inhibiting their production by inhibiting cyclooxygenases. Less is understood about the catabolism and degradation of PGs. We carried out a kinetic analysis of IL-10-induced gene expression in primary human MF in order to gain insight into mechanisms that underlie the anti-inflammatory actions of IL-10. This analysis revealed transient induction of genes encoding transcription factors followed by sustained elevation or suppression of gene expression. Several potential mediators of IL-10 antiinflammatory effects were identified, including prostaglandin dehydrogenase (PGDH), the major enzyme involved in the degradation of PGEs.

Methods Cells and reagents IL-10 was purchased from R&D Systems, Minneapolis, MN. Human peripheral blood mononuclear cells (PBMC) were isolated from venous blood of 10 independent healthy donors by centrifugation on a Ficoll density gradient (Gibco-BRL, Gaithersburg, MD). Monocytes were obtained from PBMCs using antiCD14 magnetic beads (Miltenyi Biotech, Auburn, CA) as previously described (Herrero et al., 2003), and were 498% pure as verified by flow cytometry. Monocytes were differentiated into MF by culturing for 2 days in RPMI 1640 medium (Gibco-RBL) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) and MCSF (20 ng/ml). Cells were stimulated with IL-10 (10 ng/ ml) for 0.5, 3, or 6 h. The viability and purity of MF was comparable in all conditions, and the effectiveness of IL10 action was confirmed for each donor by measuring IL-10 inhibition of LPS-induced TNFa production in parallel cultures. Cells of questionable quality were excluded from further gene expression analysis.

col (www.affymetrix.com/support/technical/manuals.affx). Biotin-labeled cRNA was hybridized to human U95Av2 oligonucleotide probe arrays (Affymetrix Incorporated, Santa Clara CA). Samples from 10 donors were pooled into two sets before hybridization. Arrays were scanned using a laser scanner (Agilent, Palo Alto, CA). All chips were scaled to an overall target intensity of 250, using Affymetrix Microarray Software v.5.0, to allow comparison between chips. Then, gene expression data were subjected to 50th percentile global normalization by using GeneSpring 6.2 software (Silicon Genetics, Redwood City, CA), as recommended by Silicon Genetics. Differentially expressed genes were selected by GeneSpring using parametric statistical group comparison (Welch t-test or Welch Anova) and the Benjamini–Hochberg false discovery rate multiple testing correction.

Real-time quantitative PCR Real-time PCR was used to confirm changes in gene expression. Briefly, total RNA was reverse-transcribed using a first strand cDNA synthesis kit (Fermentas, Hanover, MD). Real-time PCR was performed using the iCycler (BioRad, Hercules, CA) and SYBR Green PCR Core Reagents kit (Applied Biosystems, Foster City, CA). Amounts of mRNA were normalized relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The sequences of primers used to determine the levels of gene expression are as follows: autotaxin (50 -ACA TTT AGT CCT GTA CTG TAT GGA A-30 -sense, 50 -TTA ACA GTT AAC AGC AAA TAA AGG C-30 -antisense), interleukin-7 (50 -ACG GAT TAG GGC ATT TGA G-30 -sense, 50 -AGA AAT AGT TTG TTG ACT GGA GC-30 -antisense), KIAA0963 (50 GGA CTG GGC ACC CAC AAG-30 -sense, 50 -GGT TTC ACG GAT TTC AAG G-30 -antisense), PGDH (50 -TGA AGC CAA ATC TTT CCA TGT GAG A-30 sense, 50 -GAA CAT ACA AAC AAG CTT ATG CCT G-30 -antisense), lamin B1 (50 -CGG AAG GGG TTC CTC AAA-30 -antisense, 50 -CCA AGA CGC ACA GTG GTT TAT-30 -antisense), GAPDH (50 -ATC AAG AAG GTG GTG AAG CA-30 -sense, 50 -GTC GCT GTT GAA GTC AGA GGA-30 -antisense), CCR7 (50 -TCC CAC AGA CTC AAA TGC TC-30 -sense, 50 -TTC CTC ACC AAG CCA AGA AG-30 -antisense), insulin-like growth factor 1 (IGF-1) (50 -CAG GAG GGA CTC TGA AAC CT-30 -sense, 50 -GGG CCT TTA TGT AAA CTG AAT AT-30 -antisense).

Gene expression analysis

Measurement of PGE2 metabolites in macrophageconditioned media

Extraction, purification, and processing of RNA was performed according to the standard Affymetrix proto-

MF were cultured in the presence or absence of PGE2 (Cayman Chemical Company, Ann Arbor, MI), the

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substrate for PGDH. PGE2 metabolites were measured by converting all of the intermediate PGE2 metabolites to a single, stable derivative bicyclo PGE2 that was quantified by enzyme immunoassay (EIA) using the bicyclo PGE EIA kit (Cayman Chemical Company, Ann Arbor, MI). All procedures were performed according to the manufacturer’s protocol.

Results Kinetics of gene induction by IL-10 in human macrophages Our goal was to analyze the dynamic pattern of IL10-inducible gene expression in human blood-derived MF. We used Affymetrix oligonucleotide microarrays to identify differences in gene expression at several time points after addition of IL-10: 30 min, 3, and 6 h. Two pools of RNA derived from 10 independent healthy donors were used. A small group of 23 genes, that corresponds to 26 Affymetrix probe sets, was transiently

Table 1.

89

induced by IL-10 by greater than 2-fold in a statistically significant manner (po0.05 by Welch t-test, Benjamini–Hochberg false discovery rate multiple testing correction) at the 30 min time point (Table 1). Many of the induced genes (9 out of 23) represent known transcriptional factors and regulators, such as EGR2, c-Fos and Jun-B. At the same time point, after 30 min of incubation with IL-10, there were no genes suppressed by IL-10 that passed statistical and 2-fold cut-off filters used for analysis. In contrast to transient induction of gene expression at 30 min, elevated expression of a comparable set of genes was observed at the 3 and 6 h time points (Fig. 1A and data not shown). At the 6 h time point, IL-10 induced the expression of 273 genes and suppressed the expression of 71 genes by greater than 2-fold in a statistically significant manner (po0.05 by Welch t-test, Benjamini–Hochberg false discovery rate multiple testing correction) (Fig. 1A and B). Subsets of the genes most highly induced and suppressed by IL10 after 6 h are shown in Tables 2 and 3 correspondingly. A small subset of the genes (o20%) induced by 6 h of IL-10 treatment remained elevated after 2 days of IL-10 induction (Park-Min et al., 2005). Interestingly,

Genes induced after 30 min stimulation of macrophages by IL-10a

Description

Common name

GeneBank

Fold induction

Signal intensityb

STAT-induced STAT inhibitor 3 Thrombomodulin Early growth response 2 Monocyte chemotactic protein-2 v-fos FBJ murine osteosarcoma viral oncogene homolog Early growth response 3 Cyclin-dependent kinase inhibitor 1A (p21, Cip1)

CIS3 SSI3 SOCS3 THRMCD141 KROX20 MCP-2 c-fos

AB004904 J02973 J04076 Y16645 V01512

94.5 6.8 6.7 4.7 4.6

1534 325 665 551 1243

Pilot CDKN1 CIP1 WAF1 CAP20 P21 MYD118GADD45BETA IL1B TTPTIS11 MT1 JUNB ETR101

 63741 U03106

3.8 3.7

86 2166

AF078077 M15330 M92843 J03910  51345 M62831 AA224832 U72649 J05008 L05072 M54915 AI806222

3.4 3.3 3.2 3.2 3.2 3.1 2.9 2.7 2.5 2.4 2.4 2.3

1677 405 8045 812 2088 1862 384 2089 397 788 1882 418

M26683 M31166 AL021154 K01383

2.3 2.1 2.1 2.0

3380 34 450 697

DKFZP566B133 protein Interleukin 1, beta Zinc finger protein homologous to Zfp-36 in mouse Metallothionein 1G jun B proto-oncogene Transcription factor ETR101 Similar to gb:X76717 H. sapiens MT-1 l mRNA BTG family, member 2 Endothelin 1 Interferon regulatory factor 1 pim-1 oncogene 30 similar to gb:X52195 5-lipoxygenase activating protein SWISS-PROT ACC P13500 Pentaxin-related gene, rapidly induced by IL-1 beta E2F transcription factor 2 Human metallothionein-I-A gene a

PC3TIS21 ET1 MAR IRF-1 PIM

Gamma.1 TSG-14 E2F-2 MT1

Genes induced by IL-1042-fold; po0.05 Welch ANOVA, Benjamini–Hochberg false discovery rate multiple testing correction. Signal intensity after IL-10 stimulation; genes were filtered for presence flag (P) in stimulated M.

b

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Fig. 1. Clusters of genes that were induced (A) or suppressed (B) after 6 h of IL-10 treatment of human blood-derived macrophages. Time points 1, 2, 3, and 4 correspond to 0 h, 30 min, 3, and 6 h of culture with IL-10 (10 ng/ml). Analysis revealed a statistically significant 42-fold induction of 273 genes and statistically significant 42-fold suppression of 71 genes.

IL-10 suppressed the basal expression of many genes that are IFN-inducible (for example, IP-10, Cig5, IFI56, ISG54, Cig49; Table 3). The complete lists of IL-10regulated genes, which partially overlap with previously reported studies on gene induction by IL-10 in human MF (Jung et al., 2004; Williams et al., 2002) and dendritic cells (Lang et al., 2002), are available as Supplemental Tables I and II.

Confirmation of microarray data with real-time PCR As an independent method to verify microarray results, real-time PCR was used to confirm the expression of 13 IL-10-inducible genes at different time points in at least three independent blood donors; representative results are shown in Fig. 2.

IL-10 induces PGDH expression and catabolism of PGs After examination of the list of IL-10-inducible genes for genes that had not previously been appreciated to mediate anti-inflammatory functions of IL-10, we chose PGDH, a key catabolic enzyme for prostaglandins, for further study. By catalyzing the conversion of the 15-hydroxyl group of prostaglandins into a keto group, this PGDH strongly reduces the biologic activity of these molecules (vanMeir et al., 1997). PGDH induction by IL-10 was confirmed in more than 10 additional independent blood donors (Fig. 3A and data not shown). PGE2 modestly induced the expression of PGDH (Fig. 3A), suggesting that PGDH mediates a previously unappreciated feedback inhibition loop that limits PGE action. Interestingly, IL-10 synergized with PGE2 in inducing PGDH expression (Fig. 3A), further supporting the notion

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Table 2.

91

Most highly induced genes after 6 h stimulation of macrophages by IL-10a

Description

Common name

GeneBank

Fold induction

Signal intensityb

DKFZp586O0118 KIAA0307 gene product Autotaxin

IR2155535 ARNT2 ATX PD-IALPHA PDNP2 IL7 G0S2 TSG-14 CIS3 SSI3 SOCS3 CD64 IGFR1 FCRI

AL049389 AB002305 L35594

90.8 57.7 54.3

366 617 2021

J04156 M69199 M31166 AB004904 M63835

42.2 30.7 29.2 26.7 18.6

178 703 525 753 3178

AB023180 X82460 X06374 U28015 U33017 Y13710

17.3 16.1 15.3 13.3 12.6 11.5

641 30 276 159 129 2857

M76665

8.4

281

AF032457

7.7

24

AB020690 X96381 AC004010 M54915 AC005390 M25393 AL023584

7.5 7.4 7.1 6.7 6.5 6.2 6.1

31 225 209 3802 1313 175 177

L20971 AF001434 L37747 U02020 AC004262 Z22970 AB018293 AB023159 D86982 X78711 D45906 AB000520

5.8 5.7 5.7 5.6 5.6 5.4 5.3 5.2 5.2 5.1 5.0 5.0

175 309 527 1399 504 982 290 39 1089 525 499 414

Interleukin 7 Putative lymphocyte G0/G1 switch gene Pentaxin-related gene, rapidly induced by IL-1 beta STAT induced STAT inhibitor 3 Fc fragment of IgG, high affinity Ia, receptor for (CD64) KIAA0963 protein 15-hydroxy prostaglandin dehydrogenase Platelet-derived growth factor alpha polypeptide Caspase 5, apoptosis-related cysteine protease Signaling lymphocytic activation molecule (SLAM) Small inducible cytokine subfamily A (Cys–Cys), member 18 Hydroxysteroid (11-beta) dehydrogenase 1 BCL2-like 11 (apoptosis facilitator) Paraneoplastic antigen MA2 ets variant gene 5 (ets-related molecule) Clone GS1-99H8 pim-1 oncogene Glutathione peroxidase 4 Protein tyrosine phosphatase dJ67K17.1 (HIV type 1 Enhancer-binding Protein 2 (Schnurri-2)) Phosphodiesterase 4B, cAMP-specific EH domain containing 1 Lamin B1 Pre-B-cell colony-enhancing factor R29368_2 CD163 antigen KIAA0750 gene product KIAA0942 protein Similar to human ankyrin 1(S08275) Glycerol kinase LIMK-2 Adaptor protein with pleckstrin homology and src homology a

KIAA0963 HPDH PDGFA ICEREL-III ICErel-III CDw150 AMAC-1 DC-CK1 PARC DCCK1 HSD11 HSD11LHSD11B BOD BIML BIM BCL2L11-PENDING MA2 MM2 KIAA0883 ERM PIM GPx-4 PTPRF HIVEP2 DPDE4 PDEIVB H-PAST PAST HPAST PBEF EMR2 M130MM130 KIAA0750 KIAA0942 KIAA0229 GK

Genes induced by IL-1045-fold; po0.05 Welch ANOVA, Benjamini–Hochberg false discovery rate multiple testing correction. Signal intensity after IL-10 stimulation; genes were filtered for presence flag (P) in stimulated M.

b

that IL-10 limits PGE action by inducing PGE catabolism. Indeed, IL-10 increased the accumulation of PGE2 metabolites in culture supernatants of MF that had been treated with PGE2 (Fig. 3B). These results suggest that IL-10 regulates PGE degradation, and that regulation of both PGE production and degradation is important in determining PGE levels.

IL-10 suppresses PGE action on macrophages To test the functional consequences of IL-10 induction of PGDH expression and PG degradation, we determined the effects of IL-10 on PGE-induced changes in IL-10 MF morphology and gene expression. Incubation of adherent MF with exogenous PGE2 resulted in detachment of most cells from the tissue culture plate;

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Table 3.

Genes suppressed after 6 h incubation of macrophages with IL-10a

Description Signal intensityb

Common name

GeneBank

Fold

Small inducible cytokine subfamily B (Cys–X–Cys), member 10 Protein tyrosine phosphatase homolog Interferon-induced protein with tetratricopeptide repeats 1 Homo sapiens cig5 mRNA Adenosine A2b receptor Interferon-induced protein with tetratricopeptide repeats 1 Butyrate response factor 2 (EGF-response factor 2) DKFZp586B0220 SDKFZp586F071 L-myc protein Interleukin 8 ISG-K54 KIAA0440 protein Butyrate response factor 2 (EGF-response factor 2) Hexokinase 2 Interferon-induced protein with tetratricopeptide repeats 4 Oxidised low-density lipoprotein (lectin-like) receptor 1 GRO3 oncogene Adenylate cyclase 9 Dual specificity phosphatase MKP-5 Regulator of G-protein signalling 2, 24kD DKFZp586I1823 Transcription factor AP-4 (activating enhancer-binding protein 4) Tumor necrosis factor (ligand) superfamily, member 8 Hypothetical protein, expressed in osteoblast L-arginine:glycine amidinotransferase Chromosome 18 open reading frame 1 P311 protein Chromosome 18 open reading frame 1 MAX-interacting protein 1 ATP-binding cassette sub-family G member 1 isoform a

INP10 IFI10 IP-10

X02530

16.7

2

LYPLYP2LOC51615 G10P1 IFNAI1 IFI56IFI56 cig5 ADORA2B G10P1 IFNAI1 IFI56IFI56 ERF2TIS11D

AF001846 M24594

10.4 9.8

4 51

AF026941 X68487 M24594

9.6 7.1 7.0

10 15 55

U07802 AL049435 AL050125 M19720 M28130 M14660 AB007900 X78992 Z46376 AF026939

6.8 6.6 6.4 6.0 5.7 5.7 5.5 5.4 4.9 4.6

167 24 16 27 117 21 9 597 26 87

AP-4

AF079167 M36821 AF036927 AB026436 L13463 AL080213 S73885

4.5 4.5 4.3 4.0 3.9 3.9 3.7

17 54 45 85 63 19 119

CD30LG CD30L GS3686 Glycine amidinotransferase C18orf1 D4S114PRO1873PTZ17 C18orf1 MXI1 WHITE1 ABC8

L09753 AB000115 S68805 AF009426 U30521 AF009425 L07648 X91249

3.6 3.6 3.5 3.4 3.1 3.1 3.1 3.0

21 113 126 33 110 42 247 59

a

LMYC MYCL IL8 G10P2 IFI54 IFI-54 KIAA0440 ERF2TIS11D HK2 cig49 LOX-1 LOX1 MIP2BSCYB3 ADCY9 MKP5 G0S8

suppression

Genes supressed by IL-1043-fold; po0.05 Welch ANOVA, Benjamini–Hochberg false discovery rate multiple testing correction. Signal intensity after IL-10 stimulation; genes were filtered for presence flag (P) in control MF.

b

the MF that remained attached lost their characteristic dendrites and assumed a rounded appearance (Fig. 4A and B). The PGE-induced detachment of MF and changes in morphology were prevented by pre-incubation of MF with IL-10 (Fig. 4C and 4D). To corroborate our morphologic observations, we used real-time PCR to examine the pattern of PGE2-inducible gene activation in MF preincubated with IL-10 for 3 h. Strikingly, induction of PGE2-inducible genes such as CCR7 and IGF1 was completely abrogated in MF by addition of IL-10 to the culture before PGE2 (Fig. 5). CCR7 up-regulation by PGE2 was blocked by IL-10 in five independent experiments using MF from different

blood donors and IGF1 induction was blocked by IL-10 in four out of five independent experiments. Thus, PGE2 activation of human MF was significantly attenuated by IL-10.

Discussion In this study, we used Affymetrix microarray technology to gain insight into dynamics of the IL-10 response in human blood-derived MF. Several studies on IL-10 transcriptional profiling have been reported that are

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IL-10

control

IL-7

IL-10

IL-10

Lamin B

control

KIAA0963

control

Autotaxin

IL-10

50 45 40 35 30 25 20 15 10 5 0

control

relative expression

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Fig. 2. Confirmation of IL-10-induced gene expression. Human macrophages were stimulated with IL-10 (10 ng/ml) for 6 h and amounts of mRNA were measured with quantitative real-time PCR and normalized relative to GAPDH. Representative results out of 13 genes tested in three independent blood donors are shown.

informative, complementary, and different from ours as they use different array technology (Williams et al., 2002), types of cells (Perrier et al., 2004), sequence of IL10 treatment relative to cell purification (Jung et al., 2004), or mouse MF (Lang et al., 2002). The IL-10 transcriptional response in our experimental model consisted of at least two consecutive waves of transcriptional activation. The first cascade, transiently induced by IL-10 at the 30 min time point, includes 23 immediate–early genes, in many cases transcriptional regulators. Although IL-10 induction of gene expression is broadly dependent on Stat3 (Lang et al., 2002), it is likely that the transcription factors induced shortly after IL-10 stimulation contributed to the second cascade of

20 relative expression

CCR7

8 6 4 2

IGF

15 10 5

IL10+PGE

IL10

IL10+PGE

PGE

0

0 IL10

Fig. 3. IL-10 induces PGDH expression synergistically with PGE2 and increases production of PGE2 degradation products. (A) Macrophages were treated with PGE2 alone (10 nM) for the indicated times or subsequently with IL-10 (10 ng/ml) that was added for 3 h. PGDH mRNA levels were measured using real-time PCR. (B) Macrophages were cultured with PGE2 and IL-10 for 8 h and accumulation of PGE metabolites was measured using ELISA.

10

control

B

control

IL10+PGE 6h

IL10

PGE 6h

IL10+PGE 3h

A

PGE 3h

control

0

Fig. 4. Morphology of macrophages attached to tissue culture plates in complete medium (A), or after 3 h of treatment with PGE2 (100 nM) (B) or IL-10 (20 ng/ml) (C). In (D), cells were preincubated with IL-10 for 3 h prior to adding PGE2 for an additional 3 h.

relative expression

50

PGE

100

120 100 80 60 40 20 0 IL10+PGE

150

control

concentration pg/ml

relative expression

bicyclo PGE

PGE

PGDH

200

Fig. 5. IL-10 blocks expression of PGE-inducible genes. mRNAs for CCR7 and IGF1 were measured with real-time PCR. Macrophages were treated with 10 ng/ml IL-10 for 3 h or left without treatment, and then stimulated with 10 nM of PGE2 for 3 h.

transcriptional activation that was apparent after 3 h of IL-10 stimulation and sustained out to the 6 h time point. This second cascade was comprised of a much larger group of genes (more than 200) representing different functional groups. The pattern of IL-10-induced suppression of genes that are constitutively expressed in MF has not been

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previously described. As one could predict, expression of inflammatory chemokines and TNF family members (IL-8, Gro3, CD30L) was suppressed. In addition, IL-10 suppressed basal expression of IFN-inducible genes, such as IP-10, IFI56, Cig5, ISG54 and Cig49. These cultures do not contain any endogenous IFNg (Hu et al., 2003; Ji et al., 2003), but it is possible that IL-10 interrupts the action of low levels of endogenous type I IFNs that have been proposed to keep MF primed or ‘‘revved up’’ for rapid and strong responses to activating stimuli (Taniguchi and Takaoka, 2001). Thus, one mechanism by which IL-10 de-activates MF may be the interruption of positive feed-forward loops and priming. PGEs are potent inflammatory factors and PGE2 is a key activator of cell surface CCR7 expression and thus migration of monocyte-derived dendritic cells to lymph nodes (Forster et al., 1999; Scandella et al., 2002). Investigation of the regulation of PGE levels has predominantly focused on mechanisms of PGE production. Our results that IL-10 induces PGDH expression and suppresses MF responses to exogenous PGE2 highlight the fact that the balance of PGE production and degradation is important for determining PGE levels, and that both production and degradation are regulated. IL-10 inhibition of CCR7 expression likely results in the suppression of CCR7-mediated cell migration. Future work will explore the in vivo (patho)physiological significance of regulation of PGE responses by IL-10.

Acknowledgments We thank Xianchun Huang and Jenny Z. Xiang from the Cornell microarray core facility for help in hybridization of Affymetrix chips. This work was supported by grants from the NIH.

Appendix A. Supplementary materials The online version of this article contains additional supplementary data. Please visit doi:10.1016/j.imbio.2005.05.003.

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