Identification of Biologically Active Chemokine Isoforms from Ascitic Fluid and Elevated Levels of CCL18/Pulmonary and Activation-regulated Chemokine in Ovarian Carcinoma
2002; Elsevier BV; Volume: 277; Issue: 27 Linguagem: Inglês
10.1074/jbc.m112275200
ISSN1083-351X
AutoresEvemie Schutyser, Sofie Struyf, Paul Proost, Ghislain Opdenakker, Geneviève Laureys, Bruno Verhasselt, Lieven Peperstraete, Ignace Van de Putte, Alessandra Saccani, Paola Allavena, Alberto Mantovani, Jo Van Damme,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoChemokines are important in leukocyte homeostasis, inflammation, angiogenesis, and metastasis. Here, the molecular diversity of chemokines present in ovarian carcinoma was studied by purifying the proteins to homogeneity from ascitic fluid. Biologically active intact CCL2 and processed CXCL8, CCL3, and CCL18 isoforms were recovered. CCL7 and CCL20 were also purified, but their levels were 10-fold lower compared with CXCL8, CCL2, and CCL3 and even 100-fold lower than the amounts of CCL18 isolated. In ascitic fluids from patients with ovarian carcinoma (n = 12), significantly higher levels of CXCL8 and CCL18 (2.0 versus0.7 ng/ml (p = 0.01) and 120 versus 44 ng/ml (p = 0.0002), respectively) were detected compared with those in nonovarian carcinoma patients (n = 12). In contrast to CXCL8, CCL18 was not inducible in carcinoma cell lines. Immunostaining showed CCL18 expression in tumor-infiltrating cells with monocyte/macrophage morphology but not in the ovarian carcinoma cells. Our data demonstrate that biochemically heterogenous but biologically active forms of several chemokines are present at different concentrations in ovarian carcinoma ascitic fluid. This points to a delicate balance of chemokines in epithelial ovarian cancer and to a potentially major role for CXCL8 and CCL18 in this tumor. Chemokines are important in leukocyte homeostasis, inflammation, angiogenesis, and metastasis. Here, the molecular diversity of chemokines present in ovarian carcinoma was studied by purifying the proteins to homogeneity from ascitic fluid. Biologically active intact CCL2 and processed CXCL8, CCL3, and CCL18 isoforms were recovered. CCL7 and CCL20 were also purified, but their levels were 10-fold lower compared with CXCL8, CCL2, and CCL3 and even 100-fold lower than the amounts of CCL18 isolated. In ascitic fluids from patients with ovarian carcinoma (n = 12), significantly higher levels of CXCL8 and CCL18 (2.0 versus0.7 ng/ml (p = 0.01) and 120 versus 44 ng/ml (p = 0.0002), respectively) were detected compared with those in nonovarian carcinoma patients (n = 12). In contrast to CXCL8, CCL18 was not inducible in carcinoma cell lines. Immunostaining showed CCL18 expression in tumor-infiltrating cells with monocyte/macrophage morphology but not in the ovarian carcinoma cells. Our data demonstrate that biochemically heterogenous but biologically active forms of several chemokines are present at different concentrations in ovarian carcinoma ascitic fluid. This points to a delicate balance of chemokines in epithelial ovarian cancer and to a potentially major role for CXCL8 and CCL18 in this tumor. pulmonary and activation-regulated chemokine fast protein liquid chromatography liver and activation-regulated chemokine monocyte chemotactic protein macrophage inflammatory protein reversed phase high pressure liquid chromatography tumor-associated macrophages regulated on activation normal T cell expressed and secreted interleukin enzyme-linked immunosorbent assay N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine phorbol 12-myristate 13-acetate antibody interferon Chemokines are small chemotactic cytokines that are structurally divided into C, CC, CXC, and CX3C subgroups according to the positioning of conserved cysteine residues (1Strieter R.M. Standiford T.J. Huffnagle G.B. Colletti L.M. Lukacs N.W. Kunkel S.L. J. Immunol. 1996; 156: 3583-3586PubMed Google Scholar). This classification corresponds only in part with a biological division of chemokines in groups that selectively attract specific subtypes of leukocytes. For example, CC chemokines are capable to activate and chemoattract multiple leukocytic cell types. Alternative attempts were made to classify chemokines as inflammatory chemokines (i.e.inducible proteins that attract leukocytes to sites of inflammation) or constitutively released homeostatic chemokines that regulate leukocyte homing in the lymphoid system (2Mantovani A. Immunol. Today. 1999; 20: 254-257Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). Besides their function in leukocyte migration, chemokines are involved in other normal or pathological processes, like hematopoiesis, angiogenesis, cancer growth, and metastasis (3Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3246) Google Scholar, 4Gerard C. Rollins B.J. Nat. Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1199) Google Scholar). This indicates that our understanding of the exact role of a number of chemokines remains limited. As a consequence, a systematic chemokine and chemokine receptor nomenclature based on protein structure rather than on function has been introduced recently (3Zlotnik A. Yoshie O. Immunity. 2000; 12: 121-127Abstract Full Text Full Text PDF PubMed Scopus (3246) Google Scholar), despite the fact that not all chemokines nor all their receptors have been identified. Indeed, for some recently cloned chemokines such as CCL18/pulmonary and activation-regulated chemokine (PARC)1 (5Hieshima K. Imai T. Baba M. Shoudai K. Ishizuka K. Nakagawa T. Tsuruta J. Takeya M. Sakaki Y. Takatsuki K. Miura R. Opdenakker G. Van Damme J. Yoshie O. Nomiyama H. J. Immunol. 1997; 159: 1140-1149PubMed Google Scholar), the regulated production and function of the natural protein has not yet been investigated in detail, and its agonistic receptor remains unknown.Chemokines are produced by a variety of cell types including leukocytes and cancer cells (6Wang J.M. Deng X. Gong W. Su S. J. Immunol. Methods. 1998; 220: 1-17Crossref PubMed Scopus (277) Google Scholar, 7Mantovani A. Vecchi A. Sozzani S. Sica A. Allavena P. Rollins B.J. Chemokines and Cancer. Humana Press, Inc., Totowa, NJ1999: 35-49Crossref Google Scholar). Tumor-derived chemokines are important for the characteristic recruitment of leukocytes, such as tumor-associated macrophages (TAM) and lymphocytes, to the tumor environment and hence contribute to the promotion of a specific host anti-tumor immune response. However, this natural cytotoxicity against tumor cells provoked by the leukocytic infiltrate is counteracted by several tumor-favoring processes mediated by chemokines. In fact, some chemotactic cytokines derived from cancer cells or tumor-associated leukocytes act as growth factors for tumor cells (6Wang J.M. Deng X. Gong W. Su S. J. Immunol. Methods. 1998; 220: 1-17Crossref PubMed Scopus (277) Google Scholar). Other chemokines promote tumor growth and metastasis by attracting endothelial cells and by causing angiogenesis or by affecting the motility or the migration of cancer cells (4Gerard C. Rollins B.J. Nat. Immunol. 2001; 2: 108-115Crossref PubMed Scopus (1199) Google Scholar, 8Müller A. Homey B. Soto H., Ge, N. Catron D. Buchanan M.E. McClanahan T. Murphy E. Yuan W. Wagner S.N. Barrera J.L. Mohar A. Verastegui E. Zlotnik A. Nature. 2001; 410: 50-56Crossref PubMed Scopus (4410) Google Scholar). Metastasis is also indirectly and in a countercurrent way, enhanced by the chemokine-induced release of matrix-degrading enzymes by the infiltrating leukocytes such as TAM (9Opdenakker G. Van Damme J. Rollins B.J. Chemokines and Cancer. Humana Press, Inc., Totowa, NJ1999: 51-69Crossref Google Scholar). In this manner, the “macrophage balance” hypothesis (10Mantovani A. Bottazzi B. Colotta F. Sozzani S. Ruco L. Immunol. Today. 1992; 13: 265-270Abstract Full Text PDF PubMed Scopus (973) Google Scholar) elegantly depicts how TAM possess the dual potential to promote or inhibit malignant cell growth and tumor progression.Ovarian carcinoma is an intriguing model for detailed investigation of the role of chemokines and tumor-associated leukocytes in neoplasia. Epithelial ovarian cancer is an important cause of death for women, since, in most of the cases, the tumor has already asymptomatically disseminated throughout the peritoneal cavity by the time that patients are diagnosed with the disease (11Negus R.P. Balkwill F.R. Rollins B.J. Chemokines and Cancer. Humana Press, Inc., Totowa, NJ1999: 193-205Crossref Google Scholar). The presence of TAM, lymphocytes, and dendritic cells has been demonstrated in the lymphoreticular infiltrate in ovarian carcinoma (12Negus R.P. Stamp G.W. Hadley J. Balkwill F.R. Am. J. Pathol. 1997; 150: 1723-1734PubMed Google Scholar, 13Melichar B. Savary C. Kudelka A.P. Verschraegen C. Kavanagh J.J. Edwards C.L. Platsoucas C.D. Freedman R.S. Clin. Cancer Res. 1998; 4: 799-809PubMed Google Scholar). In addition, mRNA for the chemokines CCL2/monocyte chemotactic protein-1 (MCP-1), CXCL8/IL-8, and CCL3/macrophage inflammatory protein-1α (MIP-1α) and, to a lesser extent, CCL4/MIP-1β and CCL5/RANTES were detected in sections of the malignant ovary tissue and in ovarian carcinoma cell lines (12Negus R.P. Stamp G.W. Hadley J. Balkwill F.R. Am. J. Pathol. 1997; 150: 1723-1734PubMed Google Scholar, 14Burke F. Relf M. Negus R. Balkwill F. Cytokine. 1996; 8: 578-585Crossref PubMed Scopus (117) Google Scholar,15Negus R.P. Stamp G.W. Relf M.G. Burke F. Malik S.T. Bernasconi S. Allavena P. Sozzani S. Mantovani A. Balkwill F.R. J. Clin. Invest. 1995; 95: 2391-2396Crossref PubMed Scopus (262) Google Scholar). Ovarian carcinoma is often accompanied by extensive ascites accumulation, and a number of cytokines have been detected in this peritoneal fluid (16Balkwill F.R. Eur. Cytokine Netw. 1994; 5: 379-385PubMed Google Scholar, 17Radke J. Schmidt D. Böhme M. Schmidt U. Weise W. Morenz J. Geburtsh. Frauenheilkd. 1996; 56: 83-87Crossref PubMed Scopus (35) Google Scholar, 18Punnonen R. Teisala K. Kuoppala T. Bennett B. Punnonen J. Cancer. 1998; 83: 788-796Crossref PubMed Scopus (56) Google Scholar). As far as chemokines are concerned, until the present only CCL2/MCP-1, CXCL8/IL-8, and CXCL12/stromal cell-derived factor-1α (15Negus R.P. Stamp G.W. Relf M.G. Burke F. Malik S.T. Bernasconi S. Allavena P. Sozzani S. Mantovani A. Balkwill F.R. J. Clin. Invest. 1995; 95: 2391-2396Crossref PubMed Scopus (262) Google Scholar, 17Radke J. Schmidt D. Böhme M. Schmidt U. Weise W. Morenz J. Geburtsh. Frauenheilkd. 1996; 56: 83-87Crossref PubMed Scopus (35) Google Scholar, 19Scotton C.J. Wilson J.L. Milliken D. Stamp G. Balkwill F.R. Cancer Res. 2001; 61: 4961-4965PubMed Google Scholar) could be demonstrated by ELISA, and the CCL2/MCP-1 and CXCL8/IL-8 levels were reported to be significantly enhanced in ascites from patients with ovarian cancer compared with nonovarian carcinoma patients (15Negus R.P. Stamp G.W. Relf M.G. Burke F. Malik S.T. Bernasconi S. Allavena P. Sozzani S. Mantovani A. Balkwill F.R. J. Clin. Invest. 1995; 95: 2391-2396Crossref PubMed Scopus (262) Google Scholar, 17Radke J. Schmidt D. Böhme M. Schmidt U. Weise W. Morenz J. Geburtsh. Frauenheilkd. 1996; 56: 83-87Crossref PubMed Scopus (35) Google Scholar). Human ascitic fluid is a rich natural source allowing us to identify molecules that may have important functions in the tumor environment and to study their molecular diversity and biological activity at the protein level. This study reports for the first time the isolation of biologically active, posttranslationally modified chemokine isoforms from the ascitic fluid from patients with ovarian carcinoma. Furthermore, we found unusually high levels of the chemokine CCL18/PARC (5Hieshima K. Imai T. Baba M. Shoudai K. Ishizuka K. Nakagawa T. Tsuruta J. Takeya M. Sakaki Y. Takatsuki K. Miura R. Opdenakker G. Van Damme J. Yoshie O. Nomiyama H. J. Immunol. 1997; 159: 1140-1149PubMed Google Scholar), alternatively designated dendritic cell-derived CC chemokine-1 (20Adema G.J. Hartgers F. Verstraten R. de Vries E. Marland G. Menon S. Foster J., Xu, Y. Nooyen P. McClanahan T. Bacon K.B. Figdor C.G. Nature. 1997; 387: 713-717Crossref PubMed Scopus (453) Google Scholar), alternative macrophage activation-associated CC chemokine-1 (21Kodelja V. Müller C. Politz O. Hakij N. Orfanos C.E. Goerdt S. J. Immunol. 1998; 160: 1411-1418PubMed Google Scholar), and MIP-4 (22Guan P. Burghes A.H.M. Cunningham A. Lira P. Brissette W.H. Neote K. McColl S.R. Genomics. 1999; 56: 296-302Crossref PubMed Scopus (45) Google Scholar). Although CCL18/PARC was not produced by the tumor cells, its levels in ascitic fluids were significantly higher in patients with ovarian carcinoma compared with nonovarian carcinoma.DISCUSSIONThe appearance of ascitic fluid due to epithelial ovarian cancer has allowed researchers to study the role of cytokines in this disease (16Balkwill F.R. Eur. Cytokine Netw. 1994; 5: 379-385PubMed Google Scholar, 17Radke J. Schmidt D. Böhme M. Schmidt U. Weise W. Morenz J. Geburtsh. Frauenheilkd. 1996; 56: 83-87Crossref PubMed Scopus (35) Google Scholar, 18Punnonen R. Teisala K. Kuoppala T. Bennett B. Punnonen J. Cancer. 1998; 83: 788-796Crossref PubMed Scopus (56) Google Scholar). In view of the accumulation of leukocytes in this peritoneal fluid, the presence of chemokines has also been investigated. Enhanced immunoreactivity for the prototypic CXC chemokine CXCL-8/IL-8 and CC chemokine CCL2/MCP-1 has been found in ascitic fluid from ovarian carcinoma patients (15Negus R.P. Stamp G.W. Relf M.G. Burke F. Malik S.T. Bernasconi S. Allavena P. Sozzani S. Mantovani A. Balkwill F.R. J. Clin. Invest. 1995; 95: 2391-2396Crossref PubMed Scopus (262) Google Scholar, 17Radke J. Schmidt D. Böhme M. Schmidt U. Weise W. Morenz J. Geburtsh. Frauenheilkd. 1996; 56: 83-87Crossref PubMed Scopus (35) Google Scholar). The substantial volumes of ascites in the tumor environment allowed us to study for the first time the molecular heterogeneity of multiple chemokines present in this pathological fluid. We purified CCL2/MCP-1 and CXCL8/IL-8 isoforms from 850 ml of ascitic fluid from a patient with ovarian carcinoma through a standard four-step purification procedure using specific ELISAs as a screening method. Mass spectrometry, NH2-terminal sequence analysis, SDS-PAGE, and immunoblotting revealed the presence of unglycosylated intact CCL2/MCP-1-(1–76). This natural CCL2/MCP-1 was clearly chemotactically active on monocytic THP-1 cells and could therefore contribute to the general recruitment and retention of TAM and lymphocytes in the malignant ovary tissue and the ascitic fluid. However, in a group of 24 patients, we were not able to demonstrate a statistically significant increase of the CCL2/MCP-1 concentrations in ovarian carcinoma ascitic fluid compared with nonovarian carcinoma ascites. Once infiltrated in the ovarian carcinoma environment, TAM selectively displayed defective mRNA and surface expression of CC chemokine receptor-2, the unique receptor for CCL2/MCP-1 (31Sica A. Saccani A. Bottazzi B. Bernasconi S. Allavena P. Gaetano B. Fei F. LaRosa G. Scotton C. Balkwill F. Mantovani A. J. Immunol. 2000; 164: 733-738Crossref PubMed Scopus (118) Google Scholar). As a consequence, TAM did not migrate anymore in response to CCL2/MCP-1. Very recently, CC chemokine receptor-1 was found to be the only CC chemokine receptor consistently expressed in the solid ovarian tumor by the infiltrating CD68+ macrophages and CD8+lymphocytes (32Scotton C. Milliken D. Wilson J. Raju S. Balkwill F. Br. J. Cancer. 2001; 85: 891-897Crossref PubMed Scopus (86) Google Scholar).Further purification and NH2-terminal sequence analysis demonstrated the presence of two CXCL8/IL-8 isoforms in the purified ascitic fluid (i.e. CXCL8/IL-8-(6–77), the isoform that is predominantly produced by PBMC (27Van Damme J. Van Beeumen J. Conings R. Decock B. Billiau A. Eur. J. Biochem. 1989; 181: 337-344Crossref PubMed Scopus (90) Google Scholar), and CXCL8/IL-8-(5–77), an as yet undescribed isoform lacking four residues at its NH2terminus). A mixture of these two natural isoforms did still chemoattract neutrophilic granulocytes at about 1 ng/ml. This is in agreement with the fact that NH2-terminally truncated forms of CXCL8/IL-8 are more active than the intact form and with the reported chemotactic potency of a natural mixture of leukocyte-derived CXCL8/IL-8-(6–77) and CXCL8/IL-8-(8–77) isoforms (28Van den Steen P.E. Proost P. Wuyts A. Van Damme J. Opdenakker G. Blood. 2000; 96: 2673-2681Crossref PubMed Google Scholar, 29Proost P. Wuyts A. Conings R. Lenaerts J.-P. Billiau A. Opdenakker G. Van Damme J. Biochemistry. 1993; 32: 10170-10177Crossref PubMed Scopus (71) Google Scholar). In addition, we found 3-fold higher CXCL8/IL-8 levels (p = 0.01) in ovarian carcinoma compared with nonovarian carcinoma ascites, in support of existing reports on significant CXCL8/IL-8 increases in the ascitic or cyst fluid of epithelial ovarian cancer patients (17Radke J. Schmidt D. Böhme M. Schmidt U. Weise W. Morenz J. Geburtsh. Frauenheilkd. 1996; 56: 83-87Crossref PubMed Scopus (35) Google Scholar,33Ivarsson K. Runesson E. Sundfeldt K. Haeger M. Hedin L. Janson P.O. Brännström M. Gynecol. Oncol. 1998; 71: 420-423Abstract Full Text PDF PubMed Scopus (67) Google Scholar). With an average CXCL8/IL-8 concentration of 2.0 ng/ml measured in the crude ascitic fluids, the natural mixture of CXCL8/IL-8 isoforms is in theory able to chemoattract neutrophilic granulocytes to the ovarian carcinoma environment. However, the cellular infiltrate in ovarian carcinoma appears to contain only few neutrophils, mainly in the lumina of blood vessels (12Negus R.P. Stamp G.W. Hadley J. Balkwill F.R. Am. J. Pathol. 1997; 150: 1723-1734PubMed Google Scholar). Therefore, if CXCL8/IL-8 is implicated in ovarian carcinoma, it is probably not predominant in the chemoattraction of neutrophils. Instead, CXCL8/IL-8 could promote angiogenesis or influence the motility of the ovarian epithelial tumor cells, expressing the CXCL8/IL-8 receptor CXCR1 (34Ivarsson K. Ekerydh A. Fyhr I.-M. Janson P.O. Brännström M. Acta Obstet. Gynecol. 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Whether CCL3/MIP-1α could play a major role in ovarian cancer biology remains to be seen, since the CCL3/MIP-1α levels were lower than 2 ng/ml in the ascitic fluids from both ovarian carcinoma and nonovarian carcinoma patients. Two other CC chemokines not previously reported in relation to ovarian carcinoma (i.e. CCL7/MCP-3 and CCL20/LARC) were recovered, albeit in minor quantities, from ovarian carcinoma ascitic fluid. CCL7/MCP-3 was originally isolated from osteosarcoma cells (36Van Damme J. Proost P. Lenaerts J.-P. Opdenakker G. J. Exp. Med. 1992; 176: 59-65Crossref PubMed Scopus (339) Google Scholar). CCL20/LARC mRNA was previously detected in pancreatic adenocarcinoma cells and in epithelial cells of the appendix and of inflamed tonsils (37Kleeff J. Kusama T. Rossi D.L. Ishiwata T. Maruyama H. Friess H. Büchler M.W. Zlotnik A. Korc M. Int. J. Cancer. 1999; 81: 650-657Crossref PubMed Scopus (128) Google Scholar, 38Tanaka Y. Imai T. Baba M. Ishikawa I. Uehira M. Nomiyama H. 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Furthermore, these levels were significantly more elevated than in nonovarian carcinoma patients (44 ng/ml; p = 0.0002). CCL18/PARC was only recently described as a T- and B-cell chemoattractant, structurally most homologous to CCL3/MIP-1α (5Hieshima K. Imai T. Baba M. Shoudai K. Ishizuka K. Nakagawa T. Tsuruta J. Takeya M. Sakaki Y. Takatsuki K. Miura R. Opdenakker G. Van Damme J. Yoshie O. Nomiyama H. J. Immunol. 1997; 159: 1140-1149PubMed Google Scholar,20Adema G.J. Hartgers F. Verstraten R. de Vries E. Marland G. Menon S. Foster J., Xu, Y. Nooyen P. McClanahan T. Bacon K.B. Figdor C.G. Nature. 1997; 387: 713-717Crossref PubMed Scopus (453) Google Scholar, 21Kodelja V. Müller C. Politz O. Hakij N. Orfanos C.E. Goerdt S. J. Immunol. 1998; 160: 1411-1418PubMed Google Scholar, 22Guan P. Burghes A.H.M. Cunningham A. Lira P. Brissette W.H. Neote K. McColl S.R. Genomics. 1999; 56: 296-302Crossref PubMed Scopus (45) Google Scholar, 40Lindhout E. Vissers J.L.M. Hartgers F.C. 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Immunol. 2001; 166: 3284-3289Crossref PubMed Scopus (62) Google Scholar,42Pardo A. Smith K.M. Abrams J. Coffman R. Bustos M. McClanahan T.K. Grein J. Murphy E.E. Zlotnik A. Selman M. J. Leukocyte Biol. 2001; 70: 610-616PubMed Google Scholar, 46Vissers J.L. Hartgers F.C. Lindhout E. Teunissen M.B. Figdor C.G. Adema G.J. J. Leukocyte Biol. 2001; 69: 785-793PubMed Google Scholar, 47Schutyser E. Struyf S. Wuyts A. Put W. Geboes K. Grillet B. Opdenakker G. Van Damme J. Eur. J. Immunol. 2001; 31: 3755-3762Crossref PubMed Scopus (51) Google Scholar). Furthermore, the protein is inducible in monocyte/macrophages by IL-4 and staphylococcal enterotoxins and is significantly enhanced in septic and rheumatoid arthritis (47Schutyser E. Struyf S. Wuyts A. Put W. Geboes K. Grillet B. Opdenakker G. Van Damme J. Eur. J. Immunol. 2001; 31: 3755-3762Crossref PubMed Scopus (51) Google Scholar). The presence of authentic CCL18/PARC protein in human ovarian carcinoma ascitic fluid was confirmed here by the isolation of substantial amounts of chemokine (1.5 μg from 50 ml), which occurred in two isoforms: intact CCL18/PARC-(1–69) and, to a lesser extent, truncated CCL18/PARC-(1–68) lacking the COOH-terminal alanine. These CCL18/PARC isoforms showed significant chemotactic activity on lymphocytes at about 1 ng/ml. The presence of significantly enhanced levels of biologically active CCL18/PARC in the ovarian carcinoma ascitic fluid could point to an important role for CCL18/PARC in the recruitment of lymphocytes to the ovarian carcinoma environment. In addition, CCL18/PARC might be involved in other processes involving tumor progression (e.g. as growth factor for the tumor cells or as a factor directly or indirectly influencing angiogenesis or metastasis). Indeed, the recent reports on the specific chemokine receptor expression in the ovarian carcinoma environment (19Scotton C.J. Wilson J.L. Milliken D. Stamp G. Balkwill F.R. Cancer Res. 2001; 61: 4961-4965PubMed Google Scholar, 32Scotton C. Milliken D. Wilson J. Raju S. Balkwill F. Br. J. Cancer. 2001; 85: 891-897Crossref PubMed Scopus (86) Google Scholar) could not take into account the yet unknown PARC receptor. The identification of a functional CCL18/PARC receptor and of a CCL18/PARC homologue in a laboratory animal would facilitate the search for other possible target cells and relevant functions of CCL18/PARC in ovarian carcinoma.With regard to the potential producer cells of CCL18/PARC, unstimulated ovarian carcinoma SW626 cells as well as stimulated ovarian and other carcinoma cell lines failed to secrete CCL18/PARC protein, although they were good cellular sources of CCL2/MCP-1, CXCL8/IL-8, and CCL20/LARC (Fig. 7; (23Schutyser E. Struyf S. Menten P. Lenaerts J.-P. Conings R. Put W. Wuyts A. Proost P. Van Damme J. J. Immunol. 2000; 165: 4470-4477Crossref PubMed Scopus (69) Google Scholar); data not shown). Thus, ovarian carcinoma cells can account for the presence of the relatively low levels of the inflammatory chemokines CXCL8/IL-8 (Fig. 7; (34Ivarsson K. Ekerydh A. Fyhr I.-M. Janson P.O. Brännström M. Acta Obstet. Gynecol. Scand. 2000; 79: 777-784Crossref PubMed Scopus (46) Google Scholar)) and CCL2/MCP-1 (12Negus R.P. Stamp G.W. Hadley J. Balkwill F.R. Am. J. Pathol. 1997; 150: 1723-1734PubMed Google Scholar) but not for the unexpectedly high concentrations of CCL18/PARC, formerly classified as a homeostatic chemokine. The lack of CCL18/PARC expression by ovarian carcinoma cells was confirmed by immunohistochemical staining of malignant ovarian tissue and of cells isolated from the ascitic fluid. The inflammatory mononuclear cells infiltrating the tumor environment were demonstrated to be the CCL18/PARC-producing cells. It is plausible that subsets of the ovarian carcinoma-infiltrating TAM and dendritic cells are a main source of ascitic CCL18/PARC. Indeed, CCL18/PARC is expressed by macrophages, mainly by alveolar macrophages in vivo and after activation of monocyte/macrophages with alternative macrophage mediators such as IL-4 and IL-10 in vitro (5Hieshima K. Imai T. Baba M. Shoudai K. Ishizuka K. Nakagawa T. Tsuruta J. Takeya M. Sakaki Y. Takatsuki K. Miura R. Opdenakker G. Van Damme J. Yoshie O. Nomiyama H. J. Immunol. 1997; 159: 1140-1149PubMed Google Scholar, 21Kodelja V. Müller C. Politz O. Hakij N. Orfanos C.E. Goerdt S. J. Immunol. 1998; 160: 1411-1418PubMed Google Scholar, 47Schutyser E. Struyf S. Wuyts A. Put W. Geboes K. Grillet B. Opdenakker G. Van Damme J. Eur. J. Immunol. 2001; 31: 3755-3762Crossref PubMed Scopus (51) Google Scholar). These alternatively activated macrophages are presumed to be immunosuppressive, since they down-modulate Th1-mediated immunity and exert Th2-associated effector functions (48Goerdt S. Orfanos C.E. Immunity. 1999; 10: 137-142Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar). In this context, IL-10-producing immunosuppressive TAM have been detected in the ascites from ovarian carcinoma patients (49Loercher A.E. Nash M.A. Kavanagh J.J. Platsoucas C.D. Freedman R.S. J. Immunol. 1999; 163: 6251-6260PubMed Google Scholar). Therefore, the elevated CCL18/PARC levels in ovarian carcinoma ascites may point to the presence of alternatively activated TAM in this tumor environment. CCL18/PARC could thus be involved in the immunosuppression of a host anti-tumor response by attracting T-lymphocytes to suppressive TAM. Furthermore
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