of February 25, 2015.This information is current as

MacrophagesActivation in Tumor-Associated

BκDefective IL-12 Production and NF-Autocrine Production of IL-10 Mediates

MantovaniAlbertoPolentarutti, Annunciata Vecchi, Jo Van Damme and

Antonio Sica, Alessandra Saccani, Barbara Bottazzi, Nadia

http://www.jimmunol.org/content/164/2/762doi: 10.4049/jimmunol.164.2.762

2000; 164:762-767; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/164/2/762.full#ref-list-1

, 14 of which you can access for free at: cites 29 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2000 by The American Association of9650 Rockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc.,

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Autocrine Production of IL-10 Mediates Defective IL-12Production and NF-kB Activation in Tumor-AssociatedMacrophages1

Antonio Sica,2* Alessandra Saccani,* Barbara Bottazzi,* Nadia Polentarutti,*Annunciata Vecchi,* Jo Van Damme,† and Alberto Mantovani* ‡

IL-12 is a central cytokine in the activation of inflammation and immunity and in the generation of Th1-type responses. Tumor-associated macrophages (TAM) from mouse and human tumors showed defective production of IL-12. Defective IL-12 productionwas associated with lack of p50/p65 NF-kB activation. TAM produced increased amounts of the immunosuppressive cytokineIL-10. Abs against IL-10 restored the defective capacity of TAM to produce IL-12. Our data suggest that during tumor growthan IL-10-dependent pathway of diversion of macrophage function can be activated into the tumor microenvironment and resultsin the promotion of the IL-101 IL-122 phenotype of TAM. Blocking IL-10, as well as other immunosuppressive cytokines presentin the tumor microenvironment, such as TGF-b, may complement therapeutic strategies aimed at activating type I antitumorimmune responses. The Journal of Immunology,2000, 164: 762–767.

T he cytokine repertoire at the tumor site is a determinantfor successful immune responses against tumors. In thisview, immunosuppressive cytokines produced by tumors

may target and paralyze immunocompetent cells and represent amajor obstacle in cancer therapy of tumor-bearing hosts. Macro-phages have the capacity to produce a high amount of cytokinesand may provide an important contribution to establish the cyto-kine profile in the tumor microenvironment. These cells representa major component of the lymphoreticular infiltrate of tumors,whose recruitment into neoplastic tissues is governed by tumor-derived chemotactic factors (1–6). Tumor-associated macrophages(TAM)3 have an ambivalent role in the regulation of tumor growthand metastasis (1, 2, 7, 8). Although fully activated macrophagescan mediate destruction of tumor cells or tissues, in many tumorsTAM promote tumor growth and dissemination (1, 2). Alterationsin the function of TAM have been described, including the defec-tive production of reactive oxygen and nitrogen intermediates andtumor cytotoxicity (1, 9–11), and the capacity to suppress specificimmunity (12). The molecular basis responsible for the functionalphenotype of TAM has not been defined.

Macrophages produce high amounts of IL-12, a proinflamma-tory and immunoregulatory heterodimeric cytokine composed oftwo covalently linked glycosylated chains of;40 (p40) and 35(p35) kDa (13). IL-12 production by macrophages is induced upon

cell activation with a variety of stimuli, including a combination ofIFN-g and LPS (13). In addition to promoting cytokine production,proliferation, and cytotoxic activity in NK and T cells, IL-12 playsan essential role in driving differentiation of uncommitted cellstoward a type I CD41 T helper phenotype (Th1) and contributes tooptimal IFN-g production, thus being critical for promotion ofcell-mediated immunity (13). Due to its biological properties,IL-12 represents an attractive agent for anticancer therapy (13),and lack of its production may be a serious impairment for effec-tive antitumor responses. In this study, we report that TAM havea drastically defective capacity to produce IL-12 and define thealterations in transcription factors and cytokine production whichunderlie this protumor phenotype. Defective IL-12 expression isdue to autocrine production of IL-10 and is paralleled by lack ofNF-kB activation. Commitment of macrophages to high produc-tion of immunosuppressive cytokines may represent a powerfulstrategy used by tumors to escape immune surveillance.

Materials and MethodsCell culture

Peritoneal elicited macrophages (PEC) and TAM were incubated in RPMI1640 medium containing 10% FCS, 2 mM glutamine, and 100 U/ml pen-icillin-streptomycin. For the different treatments (stated in the text), weused the following concentrations: IFN-g, 100 U/ml and LPS, 100 ng/ml.

Preparation of PEC and TAM

Male 8-wk-old C57 black mice were obtained from Charles River BreedingLaboratories (Calco, Italy). Mice were inoculated intramuscularly in theleft hind limb with a total of 105 tumor cells. Tumor take was monitoredand diameters of growing tumors were measured in centimeters twice aweek by caliper. PEC and TAM were obtained 3 wk after tumor cell in-jection. TAM were isolated as described previously (14): briefly, solidtumors were disaggregated by exposure to 0.3% collagenase (Sigma, St.Louis, MO) for 40 min at 37°C; 1503 106 cells in 20 ml of RPMI 1640were seeded in 140-mm petri dishes (Cel Cult; Sterilin, Feltham, U.K.),and, after 2 h of incubation, nonadherent cells were vigorously washed.Adherent cells were.95% macrophages as assessed by morphologic andfunctional criteria. Contaminating cells were tumor cells, unidentifiedsmall mononuclear cells, and polymorphonuclear leukocytes. All culturereagents contained less than 0.125 endotoxin unit/ml as checked byLimu-lus amebocyte lysate assay (BioWhittaker, Walkersville, MD). Peritonealexudate cells were obtained from mice injected 4 days previously with 1 ml

*Istituto di Ricerche Farmacologiche “Mario Negri,” Milan, Italy;‡Department ofBiotechnology, University of Brescia, Brescia, Italy; and†Rega Institute for MedicalResearch, University of Leuven, Leuven, Belgium

Received for publication June 7, 1999. Accepted for publication November 4, 1999.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby markedadvertisementin accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by the Associazione Italiana Ricerca sul Cancro, IstitutoSuperiore di Sanita, Italy-United States of America Project on Therapy of Tumors,and Ministero Universita Ricerca Scientifica e Tecnologica Rome (Italy).2 Address correspondence and reprint requests to Dr. Antonio Sica, Istituto diRicerche Farmacologiche “Mario Negri,” via Eritrea 62, 20157 Milan, Italy. E-mailaddress: Sica@irfmn.mnegri.it3 Abbreviations used in this paper: TAM, tumor-associated macrophages; PEC, peri-toneal-elicited macrophages; MCA, 3-methylcolanthrene; NPEC, normal PEC;TPEC, tumor-bearing PEC.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00

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of 3% thyoglicollate medium (Difco, Detroit, MI) as described previously(14). A total of 603 106 macrophages was seeded in 20 ml of RPMI 1640(BioWhittaker) supplemented with 2 mM glutamine (Seromed, Munich,Germany) and 20 mM HEPES (Merck, Darmstadt, Germany) in 140-mmpetri dishes (Cel Cult; Sterilin), and, after 1 h of incubation, nonadherentcells were thoroughly washed off with jets of medium. Monolayers were.95% macrophages as assessed by morphologic and functional criteria.

Cytokines and Abs

Murine recombinant IFN-g was kindly donated by Dr. G. Garotta (Hoff-mann-LaRoche, Basilea, Switzerland). LPS (Escherichia coli055:B5) waspurchased from Difco. Rat Abs to mouse IL-10 SXC.1 (15), HB10739, andrat Abs to mouse IL-10 JES5-2A5.11 (16), HB10958, were purchased fromAmerican Type Culture Collection (Manassas, VA). Determination of mu-rine IL-12 and murine IL-10 supernatants was conducted by using ELISAkits purchased from Benfer-Sheller, Keystone Laboratories (Milan, Italy).

Northern blot analysis

Cells were cultured in medium alone or supplemented with the indicatedagents, and total RNA was purified as described (17). Ten micrograms oftotal RNA from each sample was electrophoresed under denaturing con-ditions, blotted onto Nytran membranes (Schleicher & Schuell, Keene,NH), and cross-linked by UV irradiation. Membranes were prehybridizedat 42°C in Hybrisol (Oncor, Gaithersburg, MD) and hybridized overnightwith 1 3 106cpm/ml of 32P-labeled probe. Membranes were then washedthree times at room temperature for 10 min in 0.23SSC [13SSC: 0.15 MNaCl, and 0.015 M sodium citrate (pH 7.0)], 0.1% SDS, and twice at 60°Cfor 20 min in 0.23SSC and 0.1% SDS before being autoradiographedusing Kodak XAR-5 films (Eastman, Rochester, NY) and intensifierscreens at280°C. cDNAs were labeled by random priming using a com-mercial kit (Boehringer Mannheim, Indianapolis, IN) and [a-32P]deoxy-cytidine 59-triphosphate (3000 Ci/mmol; Amersham, ArlingtonHeights, IL).

EMSA analysis

For EMSA nuclear proteins were prepared as follows: the cellular pelletwas resuspended in 10–20 times its volume in buffer A (lysis buffer): 50mM KCl, 0.5% Nonidet P-40, 25 mM HEPES (pH 7.8), 1 mM PMSF, 10mg/ml leupeptin, 20mg/ml aprotinin, and 100mM DTT and subsequentlyincubated for 5 min in ice. Cells were collected by centrifugation at 2000rpm, and the supernatant was decanted. The nuclei were washed in bufferA without Nonidet P-40, collected at 2000 rpm, and resuspended 5 min inbuffer B (extraction buffer): 500 mM KCl, 25 mM HEPES (pH 7.8), 10%glycerol, 1 mM PMSF, 10mg/ml leupeptin, 20mg/ml aprotinin, and 100mM DTT for 5 min in ice. The samples were subsequently frozen andthawed (twice) by dry ice and a 37°C water bath, rotated 20 min at 4°C, andcentrifuged at 14,000 rpm for 20 min. The clear supernatant was collectedand the proteins were dialyzed for 4 h against buffer C (dialysis buffer): 50mM KCl, 25 mM HEPES (pH 7.8), 10% glycerol, 1 mM PMSF, 10mg/mlleupeptin, 20mg/ml aprotinin, and 100mM DTT. The nuclear proteinswere incubated with radiolabeled DNA probes in a 20-ml reaction mixturecontaining 20 mM Tris (pH 7.5), 60 mM KCl, 2 mM EDTA, 0.5 mM DTT,1 mg of poly(dI-dC), and 4% Ficoll. Nucleoprotein complexes were re-solved by electrophoresis on 5% nondenaturing polyacrylamide gels in0.53 Tris-borate-EDTA buffer at 12 V/cm for 2 h at room temperature.Dried gels were exposed to Kodak XAR-5 film (Eastman) at270°C withintensifying screens. Oligonucleotides were purchased from Life Technol-ogies (Milan, Italy) and were end labeled using Klenow enzyme and[a-32P]deoxycytidine 59-triphosphate;;1 ng of labeled DNA was used ina standard EMSA reaction. The following antisera were used in supershiftanalysis: 1157, raised against the Rel homology domain containing resi-dues 339–357of human p50 and also recognizing the murine p50 (18);1226, raised against a peptide containing the C-terminal 14 residues ofhuman and murine p65 (18); anti-PU.1 (SC-352X) waspurchased fromSanta Cruz Biotechnology (Santa Cruz, CA). Double-strand oligonucleotidesequences used in EMSA are also reported here: 59- GGAGGGAGGAACTTCTTAAAATT-39 region A IL-12 p40 (nt2148 to2125); 39-CTCCCTCCTTGAAGAATTTTAAG-59; 59-GTTCCCCCAGAATGTTTT-39 region BIL-12 p40 (nt 2125 to 2110); 39- AAGGGGGTCTTACAAAAG-59; 59-GATCCAGAGGGGACTTTCCGAGAGGC-39HIV-1 kB; and 39-GTCTCCCCTGAAAGGCTCTCCGCTAG-59.

Densitometry

Densitometric analysis was performed with a scanning densitometerGS300 (Hoefer Scientific Instruments, San Francisco, CA).

ResultsDefective IL-12 expression in TAM

We implanted s.c. the 3-methylcolanthrene (MCA)-derived fibro-sarcoma MN/MCA-1 in C57B1/6 mice (1). Three weeks after tu-mor implant, TAM and PEC were obtained as described (14) andcultured for 18 h in the presence or absence of IFN-g (100 U/ml)and LPS (100 ng/ml). Supernatants were collected and assayed byELISA for IL-12 production. As shown in Fig. 1A, supernatantsfrom normal PEC (NPEC) and PEC from tumor-bearing mice(TPEC) contained high levels of IL-12 (;400 pg/ml). In contrast,

FIGURE 1. Defective IL-12 expression in TAM.A, After isolation ofTAM, NPEC and TPEC, cells were incubated for 18 h as indicated, andsupernatants were collected and assayed by ELISA to evaluate IL-12 pro-tein levels. Samples were determined in triplicate and values are the av-erage (6SD) of three separate experiments.B, Analysis of the levels ofexpression ofIL-12 p40 mRNA.C, Analysis of the levels of expression ofIL-12 p35 mRNA. Data inA–Care representative of three different exper-iments. Ten micrograms of total RNA was used in Northern blot. In eachexperiment, TAM, TPEC, and NPEC were obtained from 15 tumor-bearingmice and from 15 control mice, respectively. R. F. ind., relative fold in-duction. Values were normalized for the levels of mRNA expression ob-served in LPS-stimulated NPEC, to which a value of 1.0 was assigned.

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IL-12 protein levels were almost undetectable in supernatantsfrom TAM.

To investigate the mechanisms underlying the lack of IL-12 se-cretion by TAM, the levels of IL-12 p40 and p35 mRNA expres-sion was evaluated by Northern blot analysis. Although the levelsof IL-12 p40 mRNA expression (Fig. 1B) were strongly induced in

NPEC upon cotreatment with IFN-g (100 U/ml) and LPS (100ng/ml) for 4 h, its expression was slightly lower in TPEC andvirtually absent in TAM. Similar results were obtained when theIL-12 p35 mRNA levels were analyzed (Fig. 1C).

FIGURE 2. Lack of IL-12 NF-kB-binding activity in TAM. A, EMSAusing nuclear extracts from TAM, NPEC, or TPEC treated as indicated for4 h was performed by using the32P-labeled oligonucleotide containing theIL-12 p40kB regulatory region of the IL-12 p40 promoter (nt2148 to2125). Data inA is representative of two different experiments. R. F. ind.,relative fold induction. Values were normalized for the DNA-binding ac-tivity observed in untreated NPEC, to which a value of 1.0 was assigned.B, Competition and supershift analysis of the IL-12 p40kB DNA/proteincomplex. Anti-p50, antiserum 1157; anti-p65, antiserum 1226; anti-PU.1;N. S., nonspecific Ab. IL-12 p40kB, 100 ng of cold oligonucleotide con-taining the IL-12 p40 promoter region nt2148 to2125. B, A total of 100ng of cold oligonucleotide containing the IL-12 p40 promoter region nt2127 to2110. HIV-kB, 100 ng of cold HIV-kB oligonucleotide; N. S.,100 ng of cold nonspecific oligonucleotide. Nuclear extracts were obtainedfrom NPEC.C, Binding of nuclear extracts from TAM, NPEC, and TPECtreated as indicated for the32P-labeled HIV-kB oligonucleotide. TAM,TPEC, and NPEC were obtained from 15 tumor-bearing mice and 15 con-trol mice, respectively.

FIGURE 3. Role of autocrine IL-10 in TAM.A–C, Overexpression ofIL-10 in TAM. A, Supernatants from TAM and PEC, stimulated for 18 has indicated, were analyzed by ELISA for IL-10 protein expression. Sam-ples were determined in triplicate and values are the average (6SD) ofthree separate experiments.B, A total of 10mg of total RNAs from TAM,NPEC, and TPEC treated as indicated for 4 h was analyzed by Northernblot for IL-10 mRNA expression. Data inA–C are representative of threedifferent experiments. R. F. ind., relative fold induction. Values were nor-malized for the levels of mRNA expression observed in LPS-stimulatedNPEC, to which a value of 1.0 was assigned.C, Reversion of IL-12-de-fective secretion by anti-IL-10 mAbs. Purified TAM were activated withIFN-g/LPS in the presence of increasing concentrations (10mg/ml and 100mg/ml) of different anti-IL-10 mAbs (Aand B) as indicated, or in thepresence of the respective control Abs (Ac and Bc). After 18 h, superna-tants were collected and the concentration of IL-12 protein was analyzedby ELISA. TAM, TPEC, and NPEC used in each experiment were obtainedfrom 15 tumor-bearing mice and 15 control mice, respectively.

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Lack of IL-12 NF-kB-binding activity in TAM

Transcriptional mechanisms leading to activation of the murineIL-12 p40 promoter have been studied (19). The NF-kB half sitelocated in the proximal IL-12 p40 promoter region (nt2148 to2125) was described to functionally interact with NF-kB proteinsand identified as an essential regulatory element for transcriptionalactivity of this promoter gene. To investigate the molecular mech-anisms responsible for the suppression ofIL-12 expression, weperformed EMSA studies by using nuclear extracts from TAM andPEC from both normal and tumor-bearing mice in the presence ofa 32P-labeled oligonucleotide (nt2148 to 2125) containing theIL-12p40 NF-kB half site (Fig. 2A). In resting cells, similar levelsof a predominant DNA/protein complex b were observed in thepresence of nuclear extracts from NPEC, TPEC, or TAM. Note-worthy, upon IFN-g/LPS cell activation, while formation of com-plex b did not undergo significant variations, two inducible DNA/protein complexes (a1 and a2) were expressed at much higherlevels in nuclear extracts from NPEC and TPEC in comparison toTAM. To identify the protein components of complexes a1 and a2,competition studies and supershift analysis were performed (Fig.

2B) in the presence of nuclear extracts from NPEC. Although coldcompetition with specific (Fig. 2B,lane 6) and nonspecific (Fig.2B, B and NS,lanes 7 and 9) cold oligonucleotides verified thespecificity of this DNA/protein interaction, a cold oligonucleotidecontaining the NF-kB binding site of the HIV-long terminal repeat(20) (Fig. 2B, lane 8) was able to compete complex a1 and a2formation, suggesting the presence of NF-kB members withinthese complexes. Supershift analysis with antisera against the p50(Fig. 2B, lane 2) and p65 (Fig. 2B,lane 3) NF-kB proteins iden-tified p50 as a component of complex a2 and the p50/p65 NF-kBheterodimer as components of complex a1. The fact that anti-p50Ab (antiserum1157) did not supershift but rather competed theDNA/protein complex formation of complexes a1 and a2 is due tothe nature of this Ab which was raised against the binding domainregion of the p50 protein (18), thus preventing its binding activity.A similar DNA-binding pattern was obtained by using a32P-la-beled HIV-kB oligonucleotide as probe (Fig. 2C), suggesting thatlack of interaction of NF-kB proteins with the IL-12 p40 NF-kBsite is not selective for this DNA element, but is likely due to lackof expression of nuclear NF-kB. Thus, the identification of

FIGURE 4. A, Expression ofIL-12 and IL-10 mRNAs in TAM from different murine tumors. Ten micrograms of total RNAs from TAM and TPECtreated as indicated for 4 h was analyzed by Northern blot forIL-12 p40 andIL-10 mRNA expression. In each group, 10mice were injected with thefollowing tumors, as indicated: Moloney sarcoma virus-derived MS2, the benzo(a)pyrene-induced mFS6 sarcoma, and its derived M4 subline, theB16 melanoma. R. F. ind., relative fold induction. Values were normalized for the levels of mRNA expression observed in LPS-stimulated TPECMS2 (left) or LPS-stimulated TPEC M4 (right), to which a value of 1.0 was assigned.B, Defective IL-12 production and high secretion of IL-10in TAM from human ovarian carcinoma. IL-12 and IL-10 proteins were determined by ELISA. Three different experiments are considered and resultsare expressed as pg/ml6 SD.

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alteration in the molecular mechanisms leading to NF-kB activa-tion may be important for the defective expression of a number ofNF-kB-dependent genes involved in inflammation and immunity,in addition toIL-12 (21).

Role of autocrine IL-10

IL-10 strongly inhibits IL-12 production by human and murinemonocytes/macrophages in response to activation by LPS (22). Wehave therefore evaluated the IL-10 protein and mRNA expressionin TAM and PEC (Fig. 3). Unlike NPEC and TPEC, unstimulatedTAM expressed appreciable levels of IL-10 protein (Fig. 3A) andmRNA (Fig. 3B) comparable to those found in LPS-stimulatedPEC. LPS-induced IL-10 expression and production (300 pg/ml) inTAM was much greater (.12-fold) than in PEC. Interestingly,although IFN-g (100 U/ml) inhibited the LPS-induced IL-10mRNA expression in PEC, TAM were insensitive to this pathwayof negative regulation (Fig. 3B). Because IL-10 inhibits NF-kBactivation and IL-12 production (13, 23), it was important to ad-dress whether IL-10 production and defective IL-12 inductionwere causally related in TAM. As shown in Fig. 3C, two blockinganti-IL-10 mAbs completely restored the capacity of TAM to pro-duce IL-12 to levels similar to those of control cells.

To define whether suppression of IL-12 production by autocrineIL-10 represents a common immunosuppressive event targetingmacrophages associated with tumors, total RNA was obtainedfrom TAM isolated from the Moloney sarcoma virus-derived MS2,the benzo(a)pyrene-induced mFS6 sarcoma, its derived M4 sub-line (24) and the B16 melanoma (25). As shown in Fig. 4A, similarto the MN/MCA1 fibrosarcoma, TAM associated from the MS2,mFS6, and M4 tumors were committed to high production ofIL-10and lack of IL-12 p40 mRNAs expression. In partial contrast,TAM associated with the B16 melanoma did not express eitherIL-12 or IL-10, whereas TPEC B16 expressed lower levels ofIL-12 mRNA and significant levels ofIL-10 mRNA. Of interest, incontrast with sarcoma cells, B16 melanoma cells secreted highamounts of IL-10 (data not shown), suggesting that melanoma-derived IL-10 may be responsible for defective activation of mac-rophages associated with this tumor. Next, to ascertain the possiblerelevance of this mechanism in human tumors, TAM were isolatedfrom solid tissue of human ovarian carcinoma (e.g., see Refs. 1 and6). Cells were cultured in vitro for 24 h, and the supernatants weretested for IL-12 and IL-10. Results show (Fig. 4B), in three sep-arate experiments, IL-12 levels in supernatants from resting andstimulated TAM were very low (undetectable and 56 3 pg/ml,respectively). In contrast, TAM constitutively secreted highamounts of IL-10 (376 9 pg/ml) further increased upon activation(95 6 15 pg/ml). In contrast, activated human monocytes secretedhigh levels of IL-12 (1326 35 pg/ml) and low levels of IL-10(36 6 8 pg/ml).

DiscussionThe results presented here show that autocrine production of IL-10is a major determinant of the inhibited production of IL-12 byTAM (13, 23). The actual relevance of this inhibitory circuit wasconfirmed in a variety of mouse tumors and, more importantly, inhuman ovarian carcinoma. TAM isolated from these tumors con-sistently expressed appreciable levels of IL-10 and almost unde-tectable levels of IL-12. This observation strongly supports theidea that commitments of macrophages to high production ofIL-10 may be a common mechanism used by tumors to interferewith immune responses and promote tumor survival.

IL-10, which was first identified as a factor produced by Th2cells, inhibits cytokine production by Th1 cells (26) and contrib-

utes to the inhibition of accessory function of macrophages anddendritic cells (27). Along with TGF-b, IL-10 is spontaneouslyproduced by a variety of tumors and is believed to play a majorrole in the induction of immunosuppression during tumor growth(28). IL-10-immunosuppressive activities may be at least in partascribed to its capability to inhibit NF-kB activity (23). NF-kBplays a central role in the control of inflammation and immunity(21) and represents an ideal target for tumors to disrupt immuneresponses. In our experiments, suppression of the NF-kB-depen-dent IL-12 p40 gene expression (19) correlated with lack of NF-kB-binding activity in nuclear extracts from TAM, whereas twodifferent anti-IL-10 mAbs fully restored IL-12 production. Thismolecular pathway most likely underlies other alterations of TAMfunctions dependent on NF-kB activation, such as defectiveinducible NO synthase activation (9, 21), and represents a severeimpairment for inflammatory and immune responses. In supportof this, IL-10 knockout mice demonstrated prolonged survivaland increased capacity to reject tumors as compared with controlmice (29).

Our data suggest that during tumor growth, an IL-10-dependentpathway of diversion of macrophage function can be activated intothe tumor microenvironment. Although it remains to be deter-mined which tumor-dependent signals are involved in promotingthe IL-101 IL-122 phenotype of TAM, blocking IL-10, as well asother immunosuppressive cytokines present in the tumor microen-vironment, such as TGF-b, may complement therapeutic strategiesaimed at activating type I antitumor immune responses.

AcknowledgmentsWe thank Dr. N. Rice (Advanced Bioscience Laboratories-Basic ResearchProgram-National Cancer Institute, Frederick, MD) for the gift of p50 andp65 antisera, Dr. G. Trinchieri (Winstar Institute of Anatomy and Biology,Philadelphia, PA) for the donation of the IL-12p40 and IL-12p35 cDNAprobes, and Dr. G. Garotta (Hoffmann-LaRoche) for the donation of mu-rine recombinant IFN-g.

References1. Mantovani, A., B. Bottazzi, F. Colotta, S. Sozzani, and L. Ruco. 1992. The origin

and function of macrophages.Immunol. Today 13:265.2. Mantovani, A. 1994. Tumor-associated macrophages in neoplastic progression: a

paradigm for the in vivo function of chemokines.Lab. Invest. 71:5.3. Bottazzi, B., N. Polentarutti, R. Acero, A. Balsari, D. Boraschi, P. Ghezzi,

M. Salmona, and A. Mantovani. 1983. Regulation of macrophage content ofneoplasms by chemoattractants.Science 220:210.

4. Rollins, B.J., and M.E. Sunday. 1991. Suppression of tumor formation in vivo byexpression of JE gene in malignant cells.Mol. Cell. Biol. 11:3125.

5. Graves, D. T., V. L. Jiang, M. J. Williamson, and A. J. Valente. 1989. Identifi-cation of monocyte chemotactic activity produced by malignant cells.Science245:1490.

6. Negus, R. P., G. W. Stamp, M. G. Relf, F. Burke, S. T. Malik, S. Bernasconi,P. Allavena, S. Sozzani, A. Mantovani, and F. R. Balkwill. 1995. The detectionand localization of monocyte chemoattractant protein-1 (MCP-1) in human ovar-ian cancer.J. Clin. Invest. 95:2391.

7. Evans, R. 1972. Macrophages in syngeneic animal tumors.Transplantation 14:468.

8. Dong, Z., R. Kumar, X. Yang, and I. J. Fidler. 1997. Macrophage-derived met-alloelastase is responsible for the generation of angiostatin in Lewis lung carci-noma.Cell 88:801.

9. DiNapoli, M. R., C. L. Calderon, and D. Lopez. 1996. The altered tumoricidalcapacity of macrophages isolated from tumor-bearing mice is related to reducedexpression of the inducible nitric oxide synthase gene.J. Exp. Med. 183:1323.

10. Pace, J.L., and S. W. Russell. 1981. Activation of mouse macrophages for tumorkilling. I. Quantitative analysis of mouse macrophages for tumor killing.J. Im-munol. 126:1863.

11. Nathan, C. 1991. Role of nitric oxide synthase in macrophage antimicrobial ac-tivity. Curr. Opin. Immunol. 3:65.

12. Varesio, L., R. B. Herberman, J. M. Gerson, and H. T. Holden. 1979. Suppressionof lymphokine production by macrophages infiltrating murine virus-induced tu-mors.Int. J. Cancer 24:97.

13. Trinchieri, G. 1995. Interleukin-12: a proinflammatory cytokine with immuno-regulatory functions that bridge innate resistance and antigen-specific adaptiveimmunity. Annu. Rev. Immunol. 13:251.

14. Bottazzi, B., N. Nobili, and A. Mantovani. 1990. Expression of c-fosproto-oncogene in tumor-associated macrophages.J. Immunol. 144:4878.

766 AUTOCRINE IL-10 INHIBITS IL-12 PRODUCTION BY TAM

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15. Mosmann, T. R., J. H. Shumacher, D. F. Fiorentino, J. Leverah, K. W. Moore,and M. W. Bond. 1990. Isolation of monoclonal antibodies specific for IL-4, IL-5,IL-6, and a new Th2-specific cytokine (IL-10), cytokine synthesis inhibitory fac-tor, by using a solid phase radioimmunoadsorbent assay.J. Immunol. 145:2938.

16. MacNeil, I. A., T. Suda, K. W. Moore, T. R. Mosmann, and A. Zlotnik. 1990.IL-10, a novel growth cofactor for mature and immature T cells.J. Immunol.145:4167.

17. Sica, A., A. Saccani, A. Borsatti, C. A. Power, T. N. C. Wells, W. Luini,N. Polentarutti, S. Sozzani, and A. Mantovani. 1997. Bacterial lipopolysacchariderapidly inhibits expression of C-C chemokine receptors in human monocytes.J. Exp. Med. 185:969.

18. Rice, N. R., M. L. MacKichan, and A. Israel. 1992. The precursor of NF-kB p50has IkB-like functions.Cell 71:243.

19. Murphy, T. L., M. G. Cleveland, P. Kulesza, J. Magram, and K. M. Murphy.1995. Regulation of interleukin-12 p40 expression through an NF-kB half-site.Mol. Cell. Biol. 15:5258.

20. Kawakami, K., C. Scheidereit, and R. G. Roeder. 1990. Identification and puri-fication of a human immunoglobulin-binding protein (NF-kB) that activates tran-scription from a human immunodeficiency virus type 1 promoter in vivo.Proc.Natl. Acad. Sci. USA 85:4700.

21. Baeuerle, P. A., and T. Henkel. 1994. Function and activation of NF-kB in theimmune system.Annu. Rev. Immunol. 12:141.

22. Mosmann, T. R. 1994. Properties and functions of interleukin-10.Adv. Immunol.56:1.

23. Wang, P., P. Wu, M. I. Siegel, R. W. Egan, and M. M. Billah. 1995. Interleukin(IL)-10 inhibits nuclear factorkB (NF-kB) activation in human monocytes: IL-10and IL-4 suppress cytokine synthesis by different mechanisms.J. Biol. Chem.270:9558.

24. Taraboletti, G., L. Perin, B. Bottazzi, A. Mantovani, R. Giavazzi, andM. Salmona. 1989. Membrane fluidity affects tumor-cell motility, invasion andlung-colonizing potential.J. Immunol. 44:707.

25. Bottazzi, B., S. Walter, D. Govoni, F. Colotta, and A. Mantovani. 1992. Mono-cyte chemotactic cytokine gene transfer modulates macrophage infiltration,growth, and susceptibility to IL-2 therapy of a murine melanoma. J. Immunol.148:1280.

26. Becker, J. C., C. Czerny, and E. B. Brocker. 1994. Maintenance of clonal anergyby endogenously produced IL-10.Int. Immunol. 6:1605.

27. Luster, U., L. Folgueira, A. Juretic, M. Zuber, N. J. Luscher, M. Heberer, andG. C. Spagnoli. 1994. The pattern of cytokine gene expression in freshly excisedhuman metastatic melanoma suggests a state of reversible anergy of tumor-in-filtrating lymphocytes.Int. J. Cancer 57:612.

28. Chouaib, S., C. Asselin-Paturel, F. Mami-Chouaib, A. Caignard, and J. Y. Blay.1997. The host-tumor immune conflict: from immunosuppression to resistanceand destruction.Immunol. Today 18:493.

29. Halak, B. K., H. C. Maguire, Jr., and E. C. Lattime. 1999. Tumor-induced in-terleukin-10 inhibits type 1 immune responses directed at a tumor antigen as wellas a non-tumor antigen present at the tumor site.Cancer Res. 59:911.

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