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CC chemokine ligand 18 (CCL18) was originally discovered as pulmonary and activation-regulated chemokine (PARC), dendritic cell (DC)-chemokine 1 (DC-CK1), alternative macrophage activation-associated CC chemokine-1 (AMAC-1), and macrophage inflammatory protein-4 (MIP-4). CCL18 primarily targets lymphocytes and immature DC, although its agonistic receptor remains unknown so far. CCL18 is mainly expressed by a broad range of monocytes/macrophages and DC. A more profound understanding of the various activation programs and functional phenotypes of these producer cells might give a better insight in the proinflammatory versus anti-inflammatory role of this CC chemokine. It is interesting that CCL18 is constitutively present at high levels in human plasma and likely contributes to the physiological homing of lymphocytes and DC and to the generation of primary immune responses. Furthermore, enhanced CCL18 production has been demonstrated in several diseases, including various malignancies and inflammatory joint, lung, and skin diseases. The lack of a rodent counterpart for human CCL18 sets all hope on primate animal models to further elucidate the importance of CCL18 in vivo. This review will address these different aspects in more detail.

The internalization and intracellular trafficking of chemokine receptors have important implications for the cellular responses elicited by chemokine receptors. The major pathway by which chemokine receptors internalize is the clathrin-mediated pathway, but some receptors may utilize lipid rafts/ caveolae-dependent internalization routes. This review discusses the current knowledge and controversies regarding these two different routes of endocytosis. The functional consequences of internalization and the regulation of chemokine receptor recycling will also be addressed. Modifications of chemokine receptors, such as palmitoylation, ubiquitination, glycosylation, and sulfation, may also impact trafficking, chemotaxis and signaling. Finally, this review will cover the internalization and trafficking of viral and decoy chemokine receptors.

The interferon (IFN)-inducible chemokines, specifically, IFN-␥-inducible protein-10 (IP-10), monokine induced by IFN-␥ (Mig), and IFN-inducible T-cell ␣-chemoattractant (I-TAC), share a unique CXC chemokine receptor (CXCR3). Recently, the highly specific membrane-bound protease and lymphocyte surface marker CD26/dipeptidyl peptidase IV (DPP IV) was found to be responsible for posttranslational processing of chemokines. Removal of NH 2 -terminal dipeptides by CD26/ DPP IV alters chemokine receptor binding and signaling, and hence inflammatory and anti-human immunodeficiency virus (HIV) activities. CD26/DPP IV and CXCR3 are both markers for Th1 lymphocytes and, moreover, CD26/DPP IV is present in a soluble, active form in human plasma. This study reports that at physiologic enzyme concentrations CD26/DPP IV cleaved 50% of I-TAC within 2 minutes, whereas for IP-10 and Mig the kinetics were 3-and 10-fold slower, respectively. Processing of IP-10 and I-TAC by CD26/ DPP IV resulted in reduced CXCR3-binding properties, loss of calcium-signaling capacity through CXCR3, and more than 10-fold reduced chemotactic potency. IntroductionChemokines constitute a family of low molecular mass proteins that regulate the directed migration of specific subclasses of leukocytes during normal and inflammatory processes. [1][2][3] The cellular specificity of chemokines is determined by the restricted expression of chemokine receptors on various leukocyte cell types. 4 Chemokines are divided into subfamilies depending on the position of the first 2 cysteines in their primary sequence. The CC subfamily, with 2 adjacent cysteines, contains more than 20 different proteins that regulate the migration of monocytes, eosinophils, basophils, B and T lymphocytes, natural killer (NK) cells, and dendritic cells. The CXC chemokine subfamily, with 2 cysteines separated by one other amino acid, contains several proteins with a Glu-Leu-Arg (ELR) motif in front of the first cysteine. These ELRCXC chemokines all attract neutrophilic granulocytes to sites of inflammation. The CXC chemokines without an ELR motif can attract monocytes and B or T lymphocytes. Three of the known non-ELRCXC chemokines, specifically, interferon-␥ (IFN-␥)-inducible protein-10 (IP-10 or CXCL10), monokine induced by IFN-␥ (Mig or CXCL9), and IFN-inducible T-cell ␣-chemoattractant (I-TAC or CXCL11) recognize a single CXC chemokine receptor (CXCR), namely CXCR3. 5-8 IP-10, Mig, and I-TAC attract monocytes and activated memory Th1, but not Th2, lymphocytes. 9-11 Furthermore, eosinophils and subclasses of B and NK cells express CXCR3. 11,12 In addition to their role in leukocyte migration, chemokines play a role in angiogenesis. [13][14][15][16] CXCR2 is an important receptor for the angiogenic activity of ELRCXC chemokines. 17,18 In contrast, the molecular mechanism underlying the angiostatic activity of the non-ELRCXC chemokines IP-10, Mig, and platelet factor 4 (PF-4 or CXCL4) is not completely understood. 15 In addition to CXCR1 and CXCR2 (both receptors for angiogenic ELRCXC ch...

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 versus 0.7 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 small chemotactic cytokines that are structurally divided into C, CC, CXC, and CX 3 C subgroups according to the positioning of conserved cysteine residues (1). 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 (2). Besides their function in leukocyte migration, chemokines are involved in other normal or pathological processes, like hematopoiesis, angiogenesis, cancer growth, and metastasis (3, 4). 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 (3), 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 (5), 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 (6, 7). Tumor-derived chemokines are important for the characteristic recruitment of leukocytes, such as tumor-associated macrophages (TAM) and lymphocytes, to the ...

Biological functions of proteins are influenced by posttranslational modifications such as on/off switching by phosphorylation and modulation by glycosylation. Proteolytic processing regulates cytokine and chemokine activities. In this study, we report that natural posttranslational citrullination or deimination alters the biological activities of the neutrophil chemoattractant and angiogenic cytokine CXCL8/interleukin-8 (IL-8). Citrullination of arginine in position 5 was discovered on 14% of natural leukocyte-derived CXCL8(1–77), generating CXCL8(1–77)Cit5. Peptidylarginine deiminase (PAD) is known to citrullinate structural proteins, and it may initiate autoimmune diseases. PAD efficiently and site-specifically citrullinated CXCL5, CXCL8, CCL17, CCL26, but not IL-1β. In comparison with CXCL8(1–77), CXCL8(1–77)Cit5 had reduced affinity for glycosaminoglycans and induced less CXCR2-dependent calcium signaling and extracellular signal-regulated kinase 1/2 phosphorylation. In contrast to CXCL8(1–77), CXCL8(1–77)Cit5 was resistant to thrombin- or plasmin-dependent potentiation into CXCL8(6–77). Upon intraperitoneal injection, CXCL8(6–77) was a more potent inducer of neutrophil extravasation compared with CXCL8(1–77). Despite its retained chemotactic activity in vitro, CXCL8(1–77)Cit5 was unable to attract neutrophils to the peritoneum. Finally, in the rabbit cornea angiogenesis assay, the equally potent CXCL8(1–77) and CXCL8(1–77)Cit5 were less efficient angiogenic molecules than CXCL8(6–77). This study shows that PAD citrullinates the chemokine CXCL8, and thus may dampen neutrophil extravasation during acute or chronic inflammation.

CC and CXC chemokines coinduced in fibroblasts and leukocytes by cytokines and microbial agents determine the number of phagocytes infiltrating into inflamed tissues. Interleukin-8/ CXCL8 and stromal cell-derived factor-1/CXCL12 significantly and dose-dependently increased the migration of monocytes, expressing the corresponding CXC chemokine receptors CXCR2 and CXCR4, toward suboptimal concentrations of the monocyte chemotactic proteins CCL2 or CCL7. These findings were confirmed using different chemotaxis assays and monocytic THP-1 cells. In contrast, the combination of two CC chemokines (CCL2 plus CCL7) or two CXC chemokines (CXCL8 plus CXCL12) did not provide synergy in monocyte chemotaxis. These data show that chemokines competing for related receptors and using similar signaling pathways do not synergize. Receptor heterodimerization is probably not essential for chemokine synergy as shown in CXCR4/CCR2 cotransfectants. It is noteworthy that CCL2 mediated extracellular signal-regulated kinase 1/2 phosphorylation and calcium mobilization was significantly enhanced by CXCL8 in monocytes, indicating cooperative downstream signaling pathways during enhanced chemotaxis. Moreover, in contrast to intact CXCL12, truncated CXCL12(3-68), which has impaired receptor signaling capacity but can still desensitize CXCR4, was unable to synergize with CCL2 in monocytic cell migration. Furthermore, AMD3100 and RS102895, specific CXCR4 and CCR2 inhibitors, respectively, reduced the synergistic effect between CCL2 and CXCL12 significantly. These data indicate that for synergistic interaction between chemokines binding and signaling of the two chemokines via their proper receptors is necessary.Tissue infiltration by leukocytes is an important phenomenon of a variety of normal as well as pathological processes, including leukocyte homing, inflammation, and cancer (Murphy et al., 2000;Strieter et al., 2006). This leukocyte recruitment is tightly regulated by the interplay between endothelial cells and leukocytes, a process in which G protein-coupled receptor (GPCR) agonists, including complement factor C5a, bacterial peptides (e.g., fMLP), and chemokines, play a central role. Chemokines have been detected during inflammation in many tissues, suggesting that most, if not all, cell types can secrete chemokines after induction by appropriate stimuli . Thus, it is likely that more than one chemoattractant is present at the site of inflammation. These coinduced chemokines may cooperate to attract leukocytes to the site of infection, thereby enhancing the outcome of an inflammatory response. There are many different ways to enhance the cell influx mediated by chemokines. One possibility is the synergistic interaction between cytokines to induce chemokines followed by subsequent cooperation

CXCR3 ligands were secreted by tissue fibroblasts and peripheral blood-derived mononuclear leukocytes in response to interferon-(IFN-) and Toll-like receptor (TLR) ligands. Subsequent purification and identification revealed the presence of truncated CXCL11 variants missing up to 6 amino acids. In combination with CD26/dipeptidyl peptidase IV, the metallo-protease aminopeptidase N (APN), identical to the myeloid cell marker CD13, rapidly processed CXCL11, but not CXCL8, to generate truncated CXCL11 forms. Truncated CXCL11 had reduced binding, signaling, and chemotactic properties for lymphocytes and CXCR3-or CXCR7-transfected cells. CD13/APN-truncated CXCL11 failed to induce an intracellular calcium increase but was still able to bind and desensitize CXCR3 for intact CXCL11 signaling. CXCL11 efficiently bound to CXCR7, but CXCL11 was not able to induce calcium signaling or ERK1/2 or Akt phosphorylation through CXCR7. CD26-truncated CXCL11 failed to attract lym-phocytes but still inhibited microvascu-lar endothelial cell (HMVEC) migration. However, further processing of CXCL11 by CD13 resulted in significant reduction of inhibition of HMVEC migration. Taken together, during inflammation or cancer, CXCL11 processing by CD13 may lead to a reduced number of tumor-infiltrating lymphocytes and in a more angiogenic environment. (Blood. 2007; 110:37-44)