Evemie Schutyser

The CC chemokine CCL20 and its receptor CCR6.

CCL20, alternatively named liver and activation-regulated chemokine (LARC), macrophage inflammatory protein-3alpha (MIP-3alpha) or Exodus-1, is the only chemokine known to interact with CC chemokine receptor 6 (CCR6), a property shared with the antimicrobial beta-defensins. The ligand-receptor pair CCL20-CCR6 is responsible for the chemoattraction of immature dendritic cells (DC), effector/memory T-cells and B-cells and plays a role at skin and mucosal surfaces under homeostatic and inflammatory conditions, as well as in pathology, including cancer and rheumatoid arthritis. In this review, the discovery, the gene and protein structure, the in vitro biological activities, the cell and inducer specific expression and the tissue distribution of CCL20 and CCR6 are discussed.

Involvement of CC chemokine ligand 18 (CCL18) in normal and pathological processes

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.

Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue inflammation

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.

Synergy between Coproduced CC and CXC Chemokines in Monocyte Chemotaxis through Receptor-Mediated Events

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.

The LD78beta isoform of MIP-1alpha is the most potent CCR5 agonist and HIV-1-inhibiting chemokine.

LD78alpha and LD78beta are 2 highly related nonallelic genes that code for different isoforms of the human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha). Two molecular forms of natural LD78beta (7.778 and 7.793 kDa) were identified from conditioned media of stimulated peripheral blood mononuclear cells. Although LD78alpha and LD78beta only differ in 3 amino acids, both LD78beta variants were 100-fold more potent chemoattractants for mouse lymphocytes than was LD78alpha. On the contrary, LD78beta was only 2-fold more efficient than LD78alpha in chemoattracting human lymphocytes and monocytes. Using CC chemokine receptor-transfected cells, both molecular forms of LD78beta proved to be much more potent than LD78alpha in inducing an intracellular calcium rise through CCR5. Compared with LD78alpha and RANTES, this preferential binding of LD78beta to CCR5 resulted in a 10- to 50-fold higher potency in inhibiting infection of peripheral blood mononuclear cells by CCR5-using (R5) HIV-1 strains. To date, LD78beta is the most potent chemokine for inhibiting HIV-1 infection, and can be considered as a potentially important drug candidate for the treatment of infection with R5 HIV-1 strains.

TLR ligands and cytokines induce CXCR3 ligands in endothelial cells: enhanced CXCL9 in autoimmune arthritis

CXC chemokines are potent attractants of neutrophil granulocytes, T cells or natural killer cells. Toll-like receptors (TLR) recognize microbial components and are also activated by endogenous molecules possibly implicated in autoimmune arthritis. In contrast to CXC chemokine ligand 8 (CXCL8), no CXC chemokine receptor 3 (CXCR3) ligand (ie CXCL9, CXCL10 and CXCL11) was induced by bacterial TLR ligands in human microvascular endothelial cells (HMVEC). However, peptidoglycan (PGN), double-stranded (ds) RNA or lipopolysaccharide (LPS) (TLR2, TLR3 or TLR4 ligands, respectively) synergized with interferon-γ (IFN-γ) at inducing CXCL9 and CXCL10. In contrast, enhanced CXCL11 secretion was only obtained when IFN-γ was combined with TLR3 ligand. Furthermore, flagellin, loxoribine and unmethylated CpG oligonucleotide (TLR5, TLR7 and TLR9 ligands, respectively) did not enhance IFN-γ-dependent CXCR3 ligand production in HMVEC. In analogy with TLR ligands, tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β), in combination with IFN-γ, synergistically induced CXCL9 and CXCL11 in HMVEC and human fibroblasts, two fundamental cell types delineating the joint cavity. Etanercept, a humanized soluble recombinant p75 TNF-receptor/IgG1Fc fusionprotein, neutralized synergistic CXCL9 production induced by TNF-α plus IFN-γ, but not synergy between IFN-γ and the TLR ligands PGN or LPS. Synovial chemokine concentrations exemplify the fysiopathological relevance of the observed in vitro chemokine production patterns. In synovial fluids of patients with spondylarthropathies (ie ankylosing spondylitis or psoriatic arthritis) or rheumatoid arthritis, significantly enhanced CXCL9, but not CXCL11 levels, were detected compared to concentrations in synovial fluids of patients with metabolic crystal-induced arthritis. Thus, CXCL9 is an important chemokine in autoimmune arthritis.

The CXC chemokine GCP-2/CXCL6 is predominantly induced in mesenchymal cells by interleukin-1beta and is down-regulated by interferon-gamma: comparison with interleukin-8/CXCL8.

Human granulocyte chemotactic protein-2 (GCP-2)/CXCL6 is a CXC chemokine that functionally uses both of the IL-8/CXCL8 receptors to chemoattract neutrophils but that is structurally most related to epithelial cell–derived neutrophil attractant-78 (ENA-78)/CXCL5. This study provides the first evidence that GCP-2 protein is, compared with IL-8, weakly produced by some sarcoma, but less by carcinoma cells, and is tightly regulated in normal mesenchymal cells. IL-1β was the predominant GCP-2 inducer in fibroblasts, chondrocytes, and endothelial cells, whereas IL-8 was equally well up-regulated in these cells by TNF-α, measles virus, or double-stranded RNA (dsRNA). In contrast, lipopolysaccharide (LPS) was a relatively better stimulus for GCP-2 versus IL-8 in fibroblasts. IFN-γ down-regulated the GCP-2 production in fibroblasts induced by IL-1β, TNF-α, LPS, or dsRNA. The kinetics of GCP-2 induction by IL-1β, LPS, or dsRNA in fibroblasts differed from those of IL-8. Freshly isolated peripheral blood mononuclear leukocytes, which are a good source of IL-8 and ENA-78, failed to produce GCP-2. However, lung macrophages and blood monocyte–derived macrophages produced GCP-2 in response to LPS. Quantitatively, secretion of GCP-2 always remained inferior to that of IL-8, despite the fact that the ELISA recognized all posttranslationally modified GCP-2 isoforms. The expression of GCP-2 was confirmed in vivo by immunohistochemistry. The patterns of producer cell types, inducers and kinetics and the quantities of GCP-2 produced, suggest a unique role for GCP-2 in physiologic and pathologic processes.

Regulated production and molecular diversity of human liver and activation-regulated chemokine/macrophage inflammatory protein-3 alpha from normal and transformed cells.

Liver and activation-regulated chemokine (LARC), also designated macrophage inflammatory protein-3α (MIP-3α), Exodus, or CCL20, is a C-C chemokine that attracts immature dendritic cells and memory T lymphocytes, both expressing CCR6. Depending on the cell type, this chemokine was found to be inducible by cytokines (IL-1β) and by bacterial, viral, or plant products (including LPS, dsRNA, and PMA) as measured by a specific ELISA. Although coinduced with monocyte chemotactic protein-1 (MCP-1) and IL-8 by dsRNA, measles virus, and IL-1β in diploid fibroblasts, leukocytes produced LARC/MIP-3α only in response to LPS. However, in myelomonocytic THP-1 cells LARC/MIP-3α was better induced by phorbol ester, whereas in HEp-2 epidermal carcinoma cells IL-1β was the superior inducer. The production levels of LARC/MIP-3α (1–10 ng/ml) were, on the average, 10- to 100-fold lower than those of IL-8 and MCP-1, but were comparable to those of other less abundantly secreted chemokines. Natural LARC/MIP-3α protein isolated from stimulated leukocytes or tumor cell lines showed molecular diversity, in that NH2- and COOH-terminally truncated forms were purified and identified by amino acid sequence analysis and mass spectrometry. In contrast to other chemokines, including MCP-1 and IL-8, the natural processing did not affect the calcium-mobilizing capacity of LARC/MIP-3α through its receptor CCR6. Furthermore, truncated natural LARC/MIP-3α isoforms were equally chemotactic for lymphocytes as intact rLARC/MIP-3α. It is concluded that in addition to its role in homeostatic trafficking of leukocytes, LARC/MIP-3α can function as an inflammatory chemokine during host defense.

Synergistic induction of CXCL9 and CXCL11 by Toll‐like receptor ligands and interferon‐γ in fibroblasts correlates with elevated levels of CXCR3 ligands in septic arthritis synovial fluids

The synovial cavity constitutes the ideal stage to study the interplay between microbial Toll‐like receptor (TLR) ligands and cytokines. Infiltrated leukocytes and synovial fibroblasts produce cytokine‐ and chemokine‐induced proteases for remodeling the extracellular matrix. The regulation of chemokine function for attraction and activation of leukocytes constitutes a key feature in host immunity and resolution of inflammation after infection. Enhanced levels of the CXC chemokine ligand (CXCL9)/monokine induced by interferon‐γ (IFN‐γ) and CXCL11/IFN‐inducible T cell α chemoattractant, two chemoattractants for activated T cells and natural killer cells, and ligands for CXC chemokine receptor 3 (CXCR3) were detected in the synovial fluid of septic arthritis compared with osteo‐ and crystal arthritis patients. In vitro, IFN‐γ and TLR3 ligation by double‐stranded RNA (dsRNA) induced the expression of CXCL9 and CXCL11 in leukocytes and skin‐muscle fibroblasts, whereas ligation of TLR2, TLR4, TLR5, and TLR9 by peptidoglycan (PGN), lipopolysaccharide (LPS), flagellin, and unmethylated CpG oligonucleotides, respectively, did not. PGN and LPS, but not unmethylated CpG oligonucleotides, even inhibited IFN‐γ‐induced CXCL9 and CXCL11 expression in leukocytes. In sharp contrast, in fibroblasts, the TLR ligands PGN, dsRNA, LPS, and flagellin synergized with IFN‐γ for the production of CXCL9 and CXCL11. Although TLR ligands stimulate leukocytes to produce CXCL8/interleukin‐8 during the early innate defense, they contribute less to the production of CXCR3 ligands, whereas fibroblasts are important sources of CXCR3 ligands. These results illustrate the complex interaction between cytokines and TLR ligands in infection.

Selective induction of CCL18/PARC by staphylococcal enterotoxins in mononuclear cells and enhanced levels in septic and rheumatoid arthritis

Chemokines are mediators of innate and acquired immunity. CCL18, also designated pulmonary and activation‐regulated chemokine (PARC), dendritic cell‐derived CC chemokine‐1 (DC‐CK1), alternative macrophage activation‐associated CC chemokine‐1 (AMAC‐1) and macrophage inflammatory protein‐4 (MIP‐4), was for the first time isolated from peripheral blood mononuclear cells (PBMC) and biochemically characterized. We found that CCL18/PARC protein is spontaneously secreted by PBMC and is selectively induced in PBMC by staphylococcal enterotoxins (SEA, SEB) and IL‐4, but not by IFN‐γ andthe CXCL8/IL‐8 inducers lipopolysaccharide (LPS) or Concanavalin A. Human fibroblasts, chondrocytes and endothelial cells did not produce CCL18/PARC in response to inflammatory mediators such as measles virus, double‐stranded RNA, LPS or IL‐1β, whereas up to 150 ng/ml of CCL2/MCP‐1 was induced under these conditions. In synovial fluids from septic and rheumatoid arthritis patients, fourfold‐enhanced CCL18/PARC levels (150 ng/ml) were detected compared to those in crystal‐induced arthritis and osteoarthritis. In septic arthritis, the synovial levels of CCL18/PARC were fivefold higher than those of CXCL8/IL‐8. Immunochemistry revealed CD68+ monocytes/macrophages as the main CCL18/PARC‐producing cell type in both PBMC and arthritic synovial tissue. In addition, CD1a+ blood dendritic cells expressed CCL18/PARC. These findings suggest that monocytic cells respond to Gram‐positive bacterial infection by the production of CCL18/PARC in the synovial cavity.