Abstract
Proteolytic processing of laminin-332 by matrix metalloproteinase (MMP)-2 and MMP-14 has been shown to yield fragments that are promigratory for epithelial cells. During acute and chronic inflammation, proteases are elaborated by neutrophils and macrophages that can degrade basement membranes. We investigated the susceptibility of laminin-332 to degradation by the following neutrophil and macrophage proteases: neutrophil elastase (NE), cathepsin G, proteinase-3, and MMPs-2, -8, -9, and -12. Protease-specific differences were seen in the capacity to cleave the individual chains of laminin-332. NE and MMP-12 showed the greatest activity toward the gamma2 chain, generating a fragment similar in size to the gamma2x fragment generated by MMP-2. The digestion pattern of laminin-332 by degranulated neutrophils was nearly identical to that generated with NE alone. Digestion by supernatants of degranulated neutrophils was blocked by an inhibitor of NE, and NE-deficient neutrophils were essentially unable to digest laminin-332, suggesting that NE is the major neutrophil-derived protease that degrades laminin-332. In vivo, laminin gamma2 fragments were found in the bronchoalveolar lavage fluid of wild-type mice treated with lipopolysaccharide, whereas that obtained from NE-deficient mice showed a different cleavage pattern. In addition, NE cleaved a synthetic peptide derived from the region of human laminin gamma2 containing the MMP-2 cleavage site, suggesting that NE may generate laminin-332 fragments that are also promigratory. Both laminin-332 fragments generated by NE digestion and NE-digested laminin gamma2 peptide were found to be chemotactic for neutrophils. Collectively, these data suggest that degradation of laminin-332 by NE generates fragments with important biological activities.
Highlights
HL29594 and the Alan A. and Edith L
Laminin-332 Chains Are Differentially Susceptible to Cleavage by Neutrophil and Macrophage Proteases—The ␥2 chain of laminin-332 is cleaved by a number of matrix metalloproteinase (MMP), leading to the production of fragments that are promigratory for epithelial cells [13]
As neutrophilic inflammation is a major contributor to the acute inflammatory response that the basement membrane encounters following initial injury, we investigated whether the individual chains of laminin-332 are susceptible to cleavage by serine proteases found in neutrophils
Summary
Reagents—Rat laminin-332 was purchased from Chemicon International (Temecula, CA). Mouse laminin-111 and human laminin-511 were purchased from Sigma. BAL samples were analyzed by immunoblotting using an antibody against the ␥2 chain as described above This experiment was performed using three mice per condition three independent times. LC-mass spectrometry was performed to verify the identity of the purified peptide by the Protein and Nucleic Acid Chemistry Laboratory at Washington University School of Medicine. The reaction was terminated by the addition of phenylmethylsulfonyl fluoride to 0.8 mM, and the digests were analyzed by HPLC and electrospray mass spectrometry (Protein and Nucleic Acid Chemistry Laboratory, Washington University). Following a 90-min incubation at 37 °C in 5% CO2, the filter was stained with Wright’s stain, and nonmigrated cells from the upper side of the filter were removed, and the number of migrated neutrophils on the underside of the filter were counted in 10 random high powered fields (ϫ400) for each of the triplicate wells.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
