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)

Interactions between chemokines and enzymes are vital in immunoregulation. Structural protein citrullination by peptidylarginine deiminase (PAD) has been associated with autoimmunity. In this report, we identified a novel naturally occurring posttranslational modification of chemokines, that is, the deimination of arginine at position 5 into citrulline of CXC chemokine ligand 10 (CXCL10) by rabbit PAD and human PAD2. Citrullination reduced (> 10-fold) the chemoattracting and signaling capacity of CXCL10 for CXC chemokine receptor 3 (CXCR3) transfectants; however, it did not affect CXCR3 binding. On T lymphocytes, though, citrullinated CXCL10 remained active but was again weaker than authentic CXCL10. PAD was also able to convert CXCL11, causing an impairment of CXCR3 signaling and T-cell activation, though less pronounced than for CXCL10. Similarly, receptor binding properties of CXCL11 were not altered by citrullination. However, deimination decreased heparin binding properties of both CXCL10 and CXCL11. Overall, chemokines are the first immune modulators reported of being functionally modified by citrullination. These data provide new structure-function dimensions for chemokines in leukocyte mobilization, disclosing an anti-inflammatory role for PAD. Additionally because citrullination has severe consequences for chemokine biology, this invites to reassess the involvement and impact of PAD and citrullinated peptides in inflammation, autoimmunity, and hematologic disorders. IntroductionChemokines are a family of chemoattractive cytokines that regulate the recruitment of leukocytes toward inflammatory sites. Chemokines also exert homeostatic properties in cell migration during development and immunosurveillance and affect angiogenesis as well as tumor growth. 1,2 Based on the pattern of conserved cysteine residues in their amino acid structure, chemokines are classified into CXC, CC, CX 3 C, and C subfamilies. 1,3,4 Chemokines exhibit redundancy in their binding and signaling capacities in that the ligands can interact with more than one chemokine receptor and vice versa. 5 CXC chemokine ligand 10 (CXCL10) or interferon-␥-inducible protein-10 (IP-10) is a potent attractant of lymphocytes and natural killer cells, recognized by CXC chemokine receptor 3 (CXCR3), a 7-transmembrane spanning G protein-coupled receptor (GPCR). 6 Interestingly, CXCL10 also exerts angiostatic properties depending on CXCR3; however, the exact mechanism is still unclarified. 7 It was also suggested that CXCL10 binds to another unidentified receptor that does not interact with other CXCR3 ligands, that is, interferon T cell ␣-chemoattractant (I-TAC/CXCL11) and monokine induced by interferon-␥ (Mig/CXCL9). 8 CXCL11, on the other hand, was found to bind a second receptor, that is, CXCR7. 9 CXCR7 was reported to be expressed in various transformed cells and tumor development of human lymphoma or carcinoma in mice was diminished by treatment with a CXCR7 antagonist. [9][10][11] Recently, a function in the coordination of cell migration was al...

The CXC chemokine IFN-+ -inducible protein-10 (IP-10/CXCL10) activates CXC chemokine receptor 3 (CXCR3) and attracts activated T cells and natural killer cells. Peripheral blood mononuclear cells (PBMC) produce low but significant amounts of IP-10/CXCL10 protein upon stimulation with double-stranded (ds) RNA, the Toll-like receptor 3 (TLR3) ligand. IFN-+ is a superior IP-10/CXCL10 inducer. The bacterial TLR4 and TLR2 ligands, LPS and peptidoglycan (PGN), inhibit IFN-+ -or dsRNA-dependent IP-10/CXCL10 production in PBMC, whereas IL-8/CXCL8 production was enhanced. In fibroblasts a different picture emerges with IFN-+ inducing moderate and dsRNA provoking strong IP-10/CXCL10 production. Furthermore, treatment of fibroblasts with IFN-+ in combination with bacterial LPS or PGN results in a synergistic production of IP-10/CXCL10 and IL-8/CXCL8. The synergistic induction of IP-10/CXCL10 in fibroblasts is reflected by significantly enhanced IP-10/CXCL10 concentrations in synovial fluids of septic compared to osteoarthritis patients to reach on average higher levels than those of IL-8/CXCL8. These high amounts of IP-10/CXCL10 produced by connective tissue fibroblasts not only attract CXCR3 expressing activated Th1 cells and natural killer cells to sites of infection but may also antagonize the CCR3 dependent attraction of Th2 lymphocytes and exert CXCR3-independent, defensin-like antibacterial activity.