Proteases released in organ culture by acute dermal inflammatory lesions produced in vivo in rabbit skin by sulfur mustard: Hydrolysis of synthetic peptide substrates for trypsin-like and chymotrypsin-like enzymes

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Abstract
The purpose of these studies was to identify some of the extracellular proteolytic enzymes associated with the development and healing of acute inflammatory lesions. Lesions were produced in the skin of rabbits by the topical application of the military vesicant, sulfur mustard (SM). Full-thickness, 1-cm2 central biopsies of the lesions were organ-cultured for one to three days, and the culture fluids were assayed for proteases with a variety of substrates. When compared to culture fluids from normal skin, the culture fluids from both developing and healing SM lesions had three to six times the levels of proteases hydrolyzing two synthetic peptide substrates: (1)t-butyloxycarbonyl-Leu-Gly-Arg-4-trifluoromethylcoumarin-7-amid (Boc-Leu-Gly-Arg-AFC, herein abbreviated LGA-AFC), and (2)N-benzoyl-phenylalanine-Β-naphthyl ester (BPN). LGA-AFC is a substrate for trypsin, plasmin, plasminogen activator, thrombin, kallikrein, and the C3 and C5 convertases; BPN is a chymotrypsin and cathepsin G substrate. The culture fluids did not consistently hydrolyze four other synthetic peptide substrates or the proteins [14C]-casein and [14C]elastin. In order to determine the likely sources of LGA-AFCase and BPNase activity, we counted the number of granulocytes (PMNs), macrophages (MNs) and activated fibroblasts in histologic sections of developing and healing SM lesions, and we measured the levels of these enzymes in serum, in culture fluids of PMN and MN peritoneal exudate cells, and in culture fluids of two fibroblast cell lines. In SM lesions, serum and fibroblasts seemed to be the major source of LGA-AFCase, and serum alone the major source of BPNase. Tissue PMNs and MNs seemed to be only minor sources. The crusts of healing lesions, which were full of dead PMNs, seemed to be a rich source of both enzymes. In the SM lesion culture fluids, whether LGA-AFC and BPN were hydrolyzed by endopeptidases or only by exopeptidases could be determined by evaluating complex formation withα-macroglobulin proteinase inhibitors (αM). Endopeptidases, but not exopeptidases, are entrapped and inhibited byαM, because an internal peptide band inαM must first be hydrolyzed before molecular rearrangement (required for proteinase inhibition) occurs. The catalytic site of endopeptidases that are entrapped and inhibited byαM is known to remain active on (and reachable by) small synthetic peptide substrates such as LGA-AFC and BPN. In sodium dodecyl sulfate-polyacrylamide gel preparations of SM lesion culture fluids, we found electrophoretic bands that both stained forΜM with specific antibody with the immunoperoxidase technique and hydrolyzed LGA-AFC and/or BPN. Thus, at least some of the SM lesion enzymes that hydrolyzed LGA-AFC and BPN were endopeptidases. These proteinases probably played a local extracellular role in the inflammatory process before they were inhibited by extravasated serum inhibitors, such asαM.

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