We identify matrix metalloproteinase (MMP)-8, the polymorphonuclear (PMN) leukocyte collagenase, as a critical mediator initiating lipopolysaccharide (LPS)-responsiveness in vivo. PMN infiltration towards LPS is abrogated in Mmp8-null mice. MMP-8 cleaves LPS-induced CXC chemokine (LIX) at Ser4∼Val5 and Lys79∼Arg80. LIX bioactivity is increased upon N-terminal cleavage, enhancing intracellular calcium mobilization and chemotaxis upon binding its cognate receptor, CXCR2. As there is no difference in PMN chemotaxis in Mmp8-null mice compared with wild-type mice towards synthetic analogues of MMP-8-cleaved LIX, MMP-8 is not essential for extravasation or cell migration in collagenous matrices in vivo. However, with biochemical redundancy between MMPs 1, 2, 9, and 13, which also cleave LIX at position 4∼5, it was surprising to observe such a markedly reduced PMN infiltration towards LPS and LIX in Mmp8-/- mice. This lack of physiological redundancy in vivo identifies MMP-8 as a key mediator in the regulation of innate immunity. Comparable results were found with CXCL8/IL-8 and CXCL5/ENA-78, the human orthologues of LIX. MMP-8 cleaves CXCL8 at Arg5-Ser6 and at Val7-Leu8 in CXCL5 to activate respective chemokines. Hence, rather than collagen, these PMN chemoattractants are important MMP-8 substrates in vivo; PMN-derived MMP-8 cleaves and activates LIX to execute an in cis PMN-controlled feed-forward mechanism to orchestrate the initial inflammatory response and promote LPS responsiveness in tissue.

IL-27 signaling directly into T cells is needed for follicular T helper cell survival, germinal center formation, and the production of T cell–dependent high-affinity antibodies in mice.

IntroductionAcute inflammation is the host response to tissue injury or infection that is characterized by the production of inflammatory mediators, culminating in the initial but transient recruitment of polymorphonuclear leukocytes (PMNs) that is followed by a prolonged macrophage accumulation. 1 However, the underlying, multifactorial mechanisms that shape the extent and kinetics of PMN and macrophage recruitment, apoptosis, and clearance remain unclear. Chemokines are an important class of chemoattractant cytokines produced locally in tissues that provide the directional cues for the movement of blood-derived leukocytes in development, homeostasis, and inflammation. 2 These potent chemoattractants are classified according to the position and spacing of their N-terminal cysteine residues, with 46 human chemokines currently divided into 4 families: C, CC, CXC, and CX 3 C. 3 The initial phase of inflammation involves a subset of CXC chemokines, which rapidly attract PMNs. 4 These PMN chemoattractants contain a conserved Glu-Leu-Arg (ELR) motif proximal to the CXC sequence, which is critical in cognate receptor binding and activation, 5 as well as PMN chemotaxis. 6 In humans, there are 7 ELR ϩ CXC chemokines: CXCL1, -2, and -3, also known as growth-related oncogenes ␣, ␤, and ␥, respectively; CXCL5/epithelial cell-derived neutrophil activating peptide-78 (ENA-78); CXCL6/granulocyte chemotactic protein-2 (GCP-2); CXCL7/neutrophil-activating peptide-2 (NAP-2); and CXCL8/interleukin-8 (IL-8).All bind the CXC-receptor (CXCR) 1; CXCL6 and -8 also signal through CXCR2. 7 Mice lack complete homologs of the 7 human ELR ϩ chemokines, having only 4: mCXCL1/ keratinocyte-derived chemokine (KC); mCXCL2/macrophage-inflammatory protein-2 (MIP-2); the more recently described mCXCL3/ dendritic cell inflammatory protein-1 (DCIP-1) 8 ; and mCXCL5/ lipopolysaccharide (LPS)-induced CXC chemokine (LIX). 9 All bind a single receptor that is homologous to human CXCR2. 10 After the initial PMN influx, the next stage of inflammation is directed in part by CC chemokines consisting of CCL2/monocyte chemoattractant protein (MCP)-1, CCL7/MCP-3, CCL8/MCP-2, and CCL13/MCP-4, which target multiple leukocyte subsets (monocytes, T lymphocytes, basophils, and eosinophils). 11 Monocytes can differentiate into macrophages whose role is to phagocytose and degrade microorganisms and foreign material and to present these antigens to initiate specific immune responses. In addition, macrophages ingest apoptotic PMNs from the inflamed site as a prelude to tissue resolution. 12 However, apoptosis alone cannot account for the entirety of the reduction in PMN numbers, because the potential for continued recruitment and replacement.Resident mast cells, macrophages, and epithelial cells have been proposed to produce the initial signals responsible for the accumulation of PMNs, eosinophils, and mononuclear cells in experimental models of inflammation by secretion of chemokines such as mCXCL1 and mCCL3. 13 Modulation of specific ELR ϩ chemokines by proteolytic ...

Secreted and membrane tethered matrix metalloproteinases (MMPs) are key homeostatic proteases regulating the extracellular signaling and structural matrix environment of cells and tissues. For drug targeting of proteases, selectivity for individual molecules is highly desired and can be met by high yield active site specificity profiling. Using the high throughput Proteomic Identification of protease Cleavage Sites (PICS) method to simultaneously profile both the prime and non-prime sides of the cleavage sites of nine human MMPs, we identified more than 4300 cleavages from P6 to P6' in biologically diverse human peptide libraries. MMP specificity and kinetic efficiency were mainly guided by aliphatic and aromatic residues in P1' (with a ~32-93% preference for leucine depending on the MMP), and basic and small residues in P2' and P3', respectively. A wide differential preference for the hallmark P3 proline was found between MMPs ranging from 15 to 46%, yet when combined in the same peptide with the universally preferred P1' leucine, an unexpected negative cooperativity emerged. This was not observed in previous studies, probably due to the paucity of approaches that profile both the prime and non-prime sides together, and the masking of subsite cooperativity effects by global heat maps and iceLogos. These caveats make it critical to check for these biologically highly important effects by fixing all 20 amino acids one-by-one in the respective subsites and thorough assessing of the inferred specificity logo changes. Indeed an analysis of bona fide MEROPS physiological substrate cleavage data revealed that of the 37 natural substrates with either a P3-Pro or a P1'-Leu only 5 shared both features, confirming the PICS data. Upon probing with several new quenched-fluorescent peptides, rationally designed on our specificity data, the negative cooperativity was explained by reduced non-prime side flexibility constraining accommodation of the rigidifying P3 proline with leucine locked in S1'. Similar negative cooperativity between P3 proline and the novel preference for asparagine in P1 cements our conclusion that non-prime side flexibility greatly impacts MMP binding affinity and cleavage efficiency. Thus, unexpected sequence cooperativity consequences were revealed by PICS that uniquely encompasses both the non-prime and prime sides flanking the proteomic-pinpointed scissile bond.

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