Seminars in Cancer Biology 14 (2004) 155160

Chemokines in the recruitment and shaping ofthe leukocyte infiltrate of tumors

Alberto Mantovani a,b,, Paola Allavena a, Silvano Sozzani c,Annunciata Vecchi a, Massimo Locati b, Antonio Sica a

a Department of Immunology and Cell Biology, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, Milan 20157, Italyb Faculty of Medicine, Centro di Eccellenza per lInnovazione Diagnostica e Terapeutica (IDET) Institute of General Pathology, University of Milan, Milan

20133, Italyc Section of General Pathology and Immunology, University of Brescia, Vle Europa 11, Brescia 25123, Italy

Abstract

Leukocytes, and macrophages in particular, are an important component of the stroma of neoplastic tissues. Tumor-associated macrophages(TAM) have the properties of a polarized M2 population and are a key component of inflammatory circuits which promote tumor growthand progression. Chemokines play a key role in the recruitment and positioning of TAM and dendritic cells in tumors and contribute toshaping their functional properties. Chemokine-recruited and positioned tumor infiltrating leukocytes are a key component of inflammatorycircuits which promote tumor progression. 2003 Elsevier Ltd. All rights reserved.

Keywords: Chemokines; Recruitment; Infiltrating leukocytes

1. Introduction

Leukocytes infiltrate neoplastic tissues and are a mani-festation of inflammation in the context of neoplastic disor-ders [1]. Mononuclear phagocytes are a major componentof the leukocyte infiltrate of tumors [2]. Tumor-associatedmacrophages (TAM) have complex, dual functions in theirinteraction with neoplastic cells, but strong evidence sug-gests that they are part of inflammatory circuits that promotetumor progression.

Macrophage are versatile cells which respond and adapt tomicroenvironmental signals. Classic macrophage activationin response to microbial agents and cytokines, interferon-(IFN) in particular, has long been recognized [3]. Morerecently, it was realized that antiinflammatory molecules,such as glucocorticoid hormones, IL-4, IL-13 and IL-10, aremore than simple inhibitors of macrophage activation, inthat they induce a distinct activation program (alternativelyactivated macrophages) [47]. In analogy with the Th1/Th2dichotomy in T cell responses, macrophages exposed toIFN and IL-4 have also been referred to as M1 and M2[8]. Available information suggests that classically activated

Corresponding author. Tel.: +39-02-39014320;fax: +39-02-39014596.

E-mail address: mantovani@marionegri.it (A. Mantovani).

type I macrophages (M1) are potent effector cells whichkill microorganisms and tumor cells and produce copiousamounts of proinflammatory cytokines. In contrast, type IImacrophages (M2) tune inflammatory responses and adap-tive Th1 immunity, scavenge debris, promote angiogenesis,tissue remodeling and repair. Recent information supportthe emerging view that TAM are a polarized type II (or M2or alternatively activated) macrophage population [4,5,8].The polarized functions of TAM integrate them in circuitsof subversion of antitumor immunity and in inflammatorymechanisms which promote tumor growth and progression.

Chemokines play a key role in the recruitment and shap-ing of the leukocyte infiltrate of tumors. Historically, tumorcells have been invaluable for the discovery and character-ization of components of the chemokine system. Here wewill review how chemokines direct migration of leukocytesin neoplastic tissue and how they contribute to shaping thefunctional properties of infiltrating cells, with a focus onTAM and tumor-associated dendritic cells (DC).

2. Chemokines in polarized macrophage populations

Components of the chemokine network are differentiallymodulated in polarized macrophages. IL-10 up-regulatesthe CC chemokine receptors CCR1, CCR2 and CCR5. In

1044-579X/$ see front matter 2003 Elsevier Ltd. All rights reserved.doi:10.1016/j.semcancer.2003.10.001

156 A. Mantovani et al. / Seminars in Cancer Biology 14 (2004) 155160

contrast, CXCR2 and CXCR4 are partially down-regulatedunder the same conditions [9]. An increase of CCR2 ex-pression is also observed in monocytes exposed to dexam-ethasone [10]. IL-4 and IL-13 do not modify the expressionof CC chemokine receptors but induce functional CXCL8(IL-8) receptors in human monocytes [11]. On the contrary,monocytes exposed to LPS or IFN down-regulated CCR1,CCR2, and CCR5 [12,13]. Similarly to what reported for DC[14], exposure of monocytes to classical pro-inflammatorysignals induces the expression of functional CCR7 and theeffect is inhibited by IL-10 (Sozzani, unpublished). Cy-tokine mediators that polarize macrophages in a type IIdirection inhibit the production of various chemokines. Pro-duction of IL-8/CCL8, RANTES/CCL5 and MIPs as wellas of the IFN-inducible chemokines IP-10/CXCL10 andMIG/CXCL9 are inhibited by IL-4 and IL-10 [15]. On thecontrary, IL-4 selectively induces eotaxin-2/CCL24 [16],CCL18 and MDC/CCL22 in macrophages and DC andthese effects are inhibited by IFN [17,18]. CCL18 is alsoup-regulated by IL-10 and antiinflammatory drugs [18,19].Therefore, differential production of chemokines which at-tract Th1 (CXCL9, CXCL10) and Th2 or T regulatory cells(CCL22) integrates type I and type II macrophages in circuitsof amplification and regulation of polarized T cell responses.

3. Chemokines involved in macrophage infiltration

TAM derive from circulating monocytic precursors andin situ proliferation is generally not an important mecha-nism that sustains the mononuclear phagocyte population,at least in human tumors [20]. Several lines of evidence,including correlation between production and infiltrationin murine and human tumors, passive immunization andgene modification, indicate that chemokines play a pivotalrole in the recruitment of monocytes in neoplastic tissues[21]. Indeed, tumors have been invaluable for the discov-ery of several members of the chemokine superfamily.Chemokines are usually classified according to their consti-tutive (e.g. CXCL12) or inducible production (e.g. CCL2,CXCL8). Tumors are generally characterized by the consti-tutive expression of chemokines belonging to the induciblerealm [22]. The molecular mechanism accounting for con-stitutive expression have been defined only for CXCL1 andinvolved NFB activation. Melanoma cells display highexpression of NF-B-inducing kinase (NIK) [23] and thisphenotype is responsible for constitutive activation of IBkinase activity and MAPK signaling cascades, as well as forconstitutive activation of NF-B [24]. This may representa general mechanism underlying constitutive expression ofinflammatory chemokines in tumors.

CCL2 is probably the most frequently found CCchemokine in tumors, since its description as a tumor-derivedchemotactic factor [25]. Human tumors shown to expressCCL2 in vivo include sarcomas, gliomas, lung tumors,carcinomas of the breast, cervix and ovary, melanomas. A

recent careful analysis of the impact of CCL2 on tumorgrowth in a non tumorigenic melanoma system revealeda biphasic effect [26]. Low-level CCL2 secretion, withphysiological accumulation of TAM, promoted tumorformation, while high CCL2 secretion resulted in massivemacrophage infiltration into the tumor mass and in itsdestruction [26]. These results in a model of melanomaprogression are consistent with the macrophage balancehypothesis [20] and emphasize the protumor potential oflevels of macrophage infiltration similar to those observedin human malignant lesions.

CCL5 is produced by breast carcinoma and melanoma[27,28]. In breast carcinoma, CCL5 expression by tumorcells correlates with a more advanced stage of disease,suggesting that CCL5 may be involved in breast cancerprogression [28,29]. A variety of other chemokines havebeen detected in neoplastic tissues as products of tumorcells or stromal elements. These include CXCL12, CXCL8,CXCL1, CXCL13, CCL17, and CCL22. CXCL1 and re-lated molecules (CXCL2, CXCL3, CXCL8, or IL-8) havebeen shown to play an important role in melanoma progres-sion [30]. They do so by direct stimulation of neoplasticgrowth, promotion of inflammation and induction of an-giogenesis. CXCL8 and related chemokines act primarilyon neutrophils. However, IL-4 and IL-13 render monocytesexquisitely sensitive to CXCL8 and CXCL1 [11]. There-fore, in the tumor microenvironment where polarized typeII T cells can be present, CXCL8 and related chemokinesmay contribute to guide the positioning and to regulatethe function of TAM. Inspite of constitutive production ofCXCL8 and related chemokines by tumor cells, neutrophilsare not a major and obvious constituent of the leukocyteinfiltrate. However, these cells, though present in minutenumbers, may play a key role in triggering and sustainingthe inflammatory cascade.

Chemokines in tumors are more than leukocyte at-tractants. Transcriptional profiling has shown that CCchemokines activate a restricted and distinct program inhuman monocytes [31]. The chemokine activated transcrip-tional profile includes matrix metalloproteases (see belowfor discussion) and cytochrome CYP1B1, involved in car-cinogenesis. Moreover, CXC chemokines with an ELRmotif stimulate angiogenesis [32]. Tumor cells express re-ceptors for chemokines [28,3335] and can respond to thesemediators with increased proliferation and survival. Finally,chemokine-driven leukocyte recruitment results in digestionof the extracellular matrix which paves the way for tumorcell leaving the primary lesions (counter-current invasion,[36]). Chemokine receptors can then guide localization ofneoplastic elements at distant anatomical sites [3335].

TAM isolated from various murine tumors and fromhuman ovarian cancer express very low level of inflam-matory chemokine receptors, CCR2 in particular [37]. Ithas been shown that primary inflammatory signals, in-cluding TNF, down-regulate CCR2 in monocytes [12](Fig. 1). Therefore, down-regulation of CCR2 may in part

A. Mantovani et al. / Seminars in Cancer Biology 14 (2004) 155160 157

Fig. 1. The role of chemokines in the immunobiology of TAM. ( ) Downregulation; ( ) upregulation. Some MMPs are induced by chemokines. VMIP-I,-II, and -III (vMIPs in the figure) are encoded by HHV8 (Kaposis sarcoma virus, KSV) and attract Th2 and T regulatory cells.

reflect the monocyte-to-macrophage transition and, in part,TNF present in the ovarian cancer microenvironment [38].Down-regulation of CCR2 may serve as a mechanism tolocalize and retain macrophages in tumors.

4. Positioning of macrophages in hypoxic areas

Uneven vascularization and hypoxia are characteristicsof neoplastic tissues which affect macrophage distributionand function. TAM accumulate preferentially in the poorlyvascularized region of tumors which are characterizedby low oxygen tension. Recent studies have shown thatmacrophage migration is suppressed in hypoxic condition[39,40] and that TAM are immobilized in avascular [41]and necrotic hypoxic areas of tumors [42]. Evidence sug-gests that in hypoxia TAM are stimulated to co-operate withtumor cells and promote angiogenesis [43]. Thus, hypoxiarepresents a stress factor that, along with other microenvi-ronmental parameters, such as low pH, low glucose levelsand high lactate levels, deeply affects the biology of TAM.Expression of the hypoxia-inducible factor-2 (HIF-2)was observed in TAM from breast carcinoma [44], andHIF-1 is produced by macrophages in hypoxia in vitro, andin avascular areas of breast carcinoma [45]. Under low oxy-gen conditions, TAM switch their metabolism to anaerobicpathways and this adaptation is achieved by the increasedexpression of hypoxia-inducible genes, such as VEGF,bFGF, and CXCL8, whose transcription is controlled by thetranscription factor HIF-1 and which stimulate angiogen-esis. The relevance that the hypoxia-dependent pathwaysplay in macrophage functions was recently demonstratedby Cramer et al. [46] using conditional knockouts of thehypoxia responsive transcription factor HIF-1.

We recently observed that hypoxia selectively up-regulatesexpression of CXCR4 in various cell types includingmacrophages [47]. Along with the report by Cramer et al.[46], our data suggest that oxygen gradient may be a critical

factor for myeloid cells migration into inflammatory sites.A relay of distinct chemokinechemokine receptor inter-actions may regulate initial recruitment, tissue infiltrationand subsequent preferential accumulation in hypoxic areas,in neoplastic and non neoplastic inflammatory sites, in amultistep navigation process [48].

5. Chemokines and adaptive antitumor immunity

As mentioned above, chemokines are more than leuko-cyte attractants. Chemokine are part of amplification andregulation systems of polarized T cell responses. Work ingene-modified mice has shown that CCL2, produced by avariety of tumors, can orient specific immunity in a Th2direction, although the exact mechanism for this actionhas not been defined [49]. It may include stimulation ofIL-10 production in macrophages. In addition to being pro-duced by neoplastic cells, CCL2 is also expressed at highlevels in TAM (A. Sica, unpublished results). Antibodyblocking experiments have provided direct evidence thatCCL2 secretion in a poorly immunogenic tumor blocks thegeneration of tumor-reactive T cells [5]. ReedSternbergcells in Hodgkins lymphoma have been shown to ex-press CCL22 (MDC) and CCL17 (TARC) [50,51]. Thesechemokines recognize CCR4 which is preferentially ex-pressed on polarized Th2 cells and on T regulatory cells(Treg) as well as on monocytes [22,52]. Interestingly, inthe same tumor, stromal cells produce CCL11 (eotaxin),which attracts eosinophils and Th2 cells. Therefore, in thishuman tumor neoplastic elements and stroma use comple-mentary tools to recruit cells associated with polarized typeII responses. In the same vein of driving into tumors po-larized Th2 cells, the oncogenic virus human herpesvirus8 (HHV8), involved in the pathogenesis of Kaposis sar-coma and hematological malignancies, encodes three CCchemokines (vMIP-I, -II, and -III) which interact as ag-onists with CCR3, CCR4 and CCR8 and, accordingly,

158 A. Mantovani et al. / Seminars in Cancer Biology 14 (2004) 155160

preferentially attract polarized type II T cells [1] and, pre-sumably, Treg cells [52]. Consistently with these in vitroobservations, Kaposis sarcoma is infiltrated by CD8+ and,to a lesser extent, CD4+ cells with a predominant typeII phenotype. Therefore HHV8 virus-encoded chemokinesrepresent a strategy to subvert effective antiviral/antitumorimmunity by favouring type 2 responses and, possibly, Tregcells.

In addition to being a target for chemokines, TAM are asource of a selected set of these mediators (CCL2, CCL22,CCL18). CCL18 was recently identified as the most abun-dant chemokine in human ovarian ascites fluid [53]. Whenthe source of CCL18 was investigated, it was tracked toTAM, with no production by ovarian carcinoma cells.CCL18 is a CC chemokine produced constitutively by im-mature DC and inducible in macrophages by IL-4, IL-13,and IL-10 [19]. Since IL-4 and IL-13 are not expressedin substantial amounts in ovarian cancer, it is likely thatIL-10, produced by tumor cells and macrophages them-selves, accounts for CCL18 production by TAM. CCL18 isan attractant for naive T cells by interacting with an uniden-tified receptor [5]. Attraction of naive T cells in a peripheralmicroenvironment dominated by type II macrophages andimmature DC may induce anergy.

DC are at least in part related to the myelomonocytic dif-ferentiation pathway and some of them can originate frommonocytes. Several reports have documented the presence ofTADC in tumors [20,54,55]. TADC are differentially local-ized in tumors. In breast cancer, it was found that immaturelangerin+ DC are interspersed in the tumor mass, whereasmore mature CD83+, DC-LAMP+ DC are confined to theperitumoral area [56]. In papillary thyroid carcinoma, TADCwere localized at the invasion front of the tumor [57]. Inter-estingly, this distribution was clearly different from that ofTAM which were evenly scattered in the tissue [57]. Morerecently, ovarian tumors were reported to be infiltrated by asubset of DC, namely plasmacytoid DC [58].

Tumor cells express many CC chemokines that arelikely involved in the recruitment of DC to the tumormass [1,55,59]. The CC chemokine MIP3/CCL20 isa potent attractant of Langerhans-type DC, but not ofmonocyte-derived DC which do not express the cognatereceptor CCR6. CCL20 has been shown to be expressedin pancreatic carcinoma, renal cancer, breast carcinomaand papillary thyroid carcinoma [56,57,60,61]. CCL20 wasexpressed by carcinoma cells as well as, in pancreatic can-cer, by TAM [56,57]. Immature DC express receptors (e.g.CCR1 and CCR5) for inflammatory chemokines and CCL5has been suggested to be a major attractant for imma-ture DC produced by papillary thyroid carcinoma [57]. Inthis tumor, hepatocyte growth factor amplifies chemokineproduction by interacting with the Met receptor [57]. Ex-pression of CXCL12/SDF-1, the main chemotactic signalfor circulating plasmacytoid DC [62] was reported to beassociated to the recruitment of this DC subset to ovariancarcinoma lesions [58].

The actual significance of TADC for human tumor pro-gression is uncertain. TADC generally show an immaturephenotype. One could speculate that immature DC maymaintain tolerance to tumor antigens and that TADC, in anal-ogy with TAM [20], may in some tumors promote tumorprogression and dissemination. Production of PGE2 and re-lease of ATP by dying cells in the tumor micro-environmentmight also contribute to the inhibition of DC maturation andIL-12 production [6365]. Finally, recent studies have re-ported that a subset of myeloid cells present in lymphoidorgans and peripheral tissues mediate immunosuppresion byexploiting the metabolism of L-arginine [66].

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Chemokines in the recruitment and shaping of the leukocyte infiltrate of tumorsIntroductionChemokines in polarized macrophage populationsChemokines involved in macrophage infiltrationPositioning of macrophages in hypoxic areasChemokines and adaptive antitumor immunityReferences