Leukocyte migration and activation is orchestrated by chemokines the cleavage of which modulates their activity and glycosaminoglycan binding and thus their functions in swelling and immunity. a family-wide investigation of MMP processing of all 14 monocyte-directed CC chemokines exposed that each is definitely exactly cleaved by one or more MMPs. By MALDI-TOF-MS 149 cleavage sites were sequenced including the 1st reported instance of CCL1 CCL16 and CCL17 proteolysis. Full-length CCL15-(1-92) and CCL23-(1-99) were cleaved within their unique 31 and 32-amino acid residue extended amino termini Balicatib respectively. Unlike other CCL chemokines that drop activity and become receptor antagonists upon MMP cleavage the prominent MMP-processed products CCL15-(25-92 28 and CCL23-(26-99) are stronger agonists in calcium flux and Transwell CC receptor transfectant and monocytic THP-1 migration assays. MMP processing of CCL16-(1-97) in its extended carboxyl terminus yields two products CCL16-(8-77) and CCL16-(8-85) with both showing unexpected enhanced glycosaminoglycan binding. Hence our study reveals for the first time that MMPs activate the long Balicatib amino-terminal chemokines CCL15 and CCL23 to potent forms that have potential to increase monocyte recruitment during inflammation. by proteases and in particular by serine proteases from neutrophils and by matrix metalloproteinases (MMPs) (8 12 13 20 Serine proteases including cathepsin G and neutrophil elastase are secreted by activated neutrophils during an inflammatory response; natural inhibitors include serpins. MMPs are an important family of extracellular endopeptidases that are up-regulated in stimulated stromal cells and leukocytes and are pathognomonic of many chronic inflammatory diseases. The activity Balicatib of MMPs is usually regulated by tissue inhibitors of metalloproteinases (TIMPs) with the net individual activities of different MMPs being both beneficial and detrimental in disease (30). In the CXC chemokine subfamily the neutrophil chemoattractants CXCL8 and CXCL5 are processed in particular by the neutrophil-specific MMP-8 (also known as Balicatib collagenase-2) to become potent receptor agonists and form a feed-forward mechanism a critical step for Balicatib neutrophil recruitment (16 27 In contrast all seven neutrophil CXC agonists in man are inactivated by macrophage-derived MMP-12 terminating the recruitment of neutrophils (21). Multiple MMPs generate potent CCR1 CCR2 and CCR5 receptor antagonists by cleaving CCL2 -7 -8 and -13 to terminate monocyte recruitment (12 13 Notably proteolysis of human CC chemokines that results in an activating cleavage is Rabbit Polyclonal to TUT1. limited to serine protease activity on CCL4 (31) CCL14 (32-34) CCL15 (20) and CCL23 (20). In an assay the 92-amino acid residue CCL15-(1-92) and the 99 amino acid residue CCL23-(1-99) neither of which is usually a potent chemoattractant in the full-length form (35 36 were processed by synovial fluid from arthritic patients to the products CCL15-(25-92) and CCL23-(19-99) that have enhanced CCR1 agonist activity (20). However despite the importance of this observation the specific proteases responsible for these cleavages could not be identified despite considerable effort. Amino-terminally truncated CCL15 and CCL23 were both identified in synovial fluid from arthritic patients at concentrations of 10-100-fold that of CCL3 and CCL5 (20) indicating that these truncated chemokines may contribute to the cellular recruitment that is observed in chronic inflammation. Herein we utilized inhibitors to identify the protease classes responsible for the activating cleavages of CCL15 in synovial fluid finding that both serine proteases and MMPs are responsible. In view of the importance of macrophage recruitment this motivated us to identify other MMP chemokine substrates. Therefore we performed a global evaluation of MMP processing of all 14 CC chemokines that are involved in monocyte recruitment. We report that MMP processing of the long amino-terminal CCL15 and CCL23 chemokines and the long carboxyl-terminal CCL16 notably by the monocyte/macrophage specific MMP-12 results in increased receptor activation or GAG binding respectively. These data thereby point to a critical role for MMPs in the promotion and regulation of monocyte recruitment. Our results implicate new feed-forward mechanisms whereby macrophage and synovial fluid proteases promote the.