Direct Effects of Neutrophil Oxidants on Elastase-Induced Extracellular Matrix Proteolysis

Oxidant species produced by human polymorphonuclear leukocytes (PMN) inactivate alpha-1-protease inhibitor and thus may indirectly enhance neutrophil elastase-induced proteolysis. It is unclear, however, if PMN-derived oxidants directly enhance proteolysis of extracellular matrix by neutrophil elastase. Matrix was produced by neonatal rat aortic smooth muscle cells and pulse-labeled with 3H-lysine to allow identification of the collagen-specific amino acid, hydroxylysine (3H-HL), and the elastin specific amino acid, desmosine (3H-DES). The smooth muscle cells were lysed, and the remaining matrix was used as a culture surface and a proteolytic substrate for intact PMN and purified neutrophil elastase. Proteolysis of collagen and elastin were quantified by chromatographic separation of the marker amino acids 3H-HL and 3H-DES, which were released into the supernatant or remained in the matrix after a 3-h incubation at 37° C The peptide, formyl-methionine-leucine-phenylalanine (FMLP), produced more rapid release of myeloperoxidase than did phorbol myristate acetate (PMA), which produced more release of and H2O2 than did FMLP. The percent release of total matrix 3H-DES in the presence of PMN + FMLP was 2.45 ± 0.19% (mean ± SE, n = 6) and with PMN + PMA it was 1.32 ± 0.1% (n = 6, p < 0.01). The release of matrix 3H-HL did not differ. Neutrophil cytoplasts, which produced and H2O2 but lacked azurophilic granules, did not significantly enhance either elastin or collagen degradation by purified neutrophil elastase (NE). Pretreatment of matrix with 250 μM, exogenous H2O2, then 10 μg of NE, released 11.0 ± 0.7% (n = 7) of 3H-DES, whereas NE alone released 5.1 ± 1.5% (n = 8, p < 0.05). Pretreatment of matrix with myeloperoxidase, H2O2, then 10 μg of NE, released 11.0 ± 0.7% (n = 7) of 3H-DES, whereas NE alone released 5.1 ± 1.5% (n = 8, p < 0.05). Pretreatment of matrix with myeloperoxidase, H2O2 and Cl− followed by addition of 5 μg of NE released 4.9 ± 0.4% (n = 6) of 3H-DES, whereas NE alone released 3.1 ± 0.3% (n = 5, p < 0.05). Although exogenously supplied H2O2 and myeloperoxidase enhanced the release of matrix elastin by neutrophil elastase, in intact PMN and cytoplasts elastin and collagen proteolysis correlated more closely with the release of azurophilic granule contents than of reactive oxygen species.