The neutrophil-specific protease membrane-type 6 matrix metalloproteinase 
(MT6-MMP)/MMP-25/leukolysin is implicated in multiple sclerosis and cancer yet 
remains poorly characterized. To characterize the biological roles of MT6-MMP, 
it is critical to identify its substrates for which only seven are currently 
known. Here, we biochemically characterized MT6-MMP, profiled its tissue 
inhibitor of metalloproteinase inhibitory spectrum, performed degradomics 
analyses, and screened 26 chemokines for cleavage using matrix-assisted laser 
desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. MT6-MMP 
processes seven each of the CXC and CC chemokine subfamilies. Notably, cleavage 
of the neutrophil chemoattractant CXCL5 activates the chemokine, thereby 
increasing its agonist activity, indicating a feed-forward mechanism for 
neutrophil recruitment. Likewise, cleavage also activated CCL15 and CCL23 to 
increase monocyte recruitment. Utilizing the proteomics approach proteomic 
identification of cleavage site specificity (PICS), we identified 286 peptidic 
cleavage sites spanning from P6 to P6' from which an unusual glutamate 
preference in P1 was identified. The degradomics screen terminal amine isotopic 
labeling of substrates (TAILS), which enriches for neo-N-terminal peptides of 
cleaved substrates, was used to identify 58 new native substrates in fibroblast 
secretomes after incubation with MT6-MMP. Vimentin, cystatin C, galectin-1, 
IGFBP-7, and secreted protein, acidic and rich in cysteine (SPARC) were among 
those substrates we biochemically confirmed. An extracellular "moonlighting" 
form of vimentin is a chemoattractant for THP-1 cells, but MT6-MMP cleavage 
abolished monocyte recruitment. Unexpectedly, the MT6-MMP-cleaved vimentin 
potently stimulated phagocytosis, which was not a property of the full-length 
protein. Hence, MT6-MMP regulates neutrophil and monocyte chemotaxis and by 
generating "eat-me" signals upon vimentin cleavage potentially increases 
phagocytic removal of neutrophils to resolve inflammation.