Over a decade ago, we found chemical evidence that collagenolysis was a concomitant finding of tissue necrosis (1, 2 ) . This collagenolysis of a normally metabolically "inert" insoluble fiber, we felt, had to proceed via the release of a collagenolytic enzyme into the extracellular space of the dermis where the collagen is located. We found that there existed in the extracellular space of rat skin an inhibitor-collagenase complex which could be activated by limited proteolysis of the inhibitor (3, 4). Recently, we have isolated and purified this enzyme after trypsin activation over 450-fold (5, 6).During the course of our studies of the chemical kinetics of necrotic wounds, we often noticed that tissue collagenolysis preceded the infiltration of the wound by polymorphonuclear leukocytes (PMN) . This observation is of some significance in view of the recent report by Lazarus et al. (7), of the collagenolytic activity of granulocytes. Further, the early studies of Spector (8), suggested that peptides produced by the proteolytic degradation of various proteins all produced leukocyte emigration if they were between 5 to 14 amino acid residues in size. Therefore, it occurred to us that possibly the degradation products of collagen that were most probably released during collagenolysis from the necrotic wound might also be involved in the PMN infiltration so characteristic of inflammation.We therefore developed an in vivo modifi-1 Supported in part by the grants-in-aid program from the National Institutes of Health (FR-00284 and AM-08168), and by a contract from O.N.R. 2 Submitted in partial fulfillment of the require-(NR-105-325). ments for the degree od M. S. in biochemistry.cation (9) of the in vitro Boyden chamber technique used by Ward (10) to study chemotaxis. Using this method, we then demonstrated that soluble collagen itself was chemotactic and that this chemotaxis of soluble collagen was destroyed by gelatinization or by incubation with bacterial collagenase ( 9 ) , but was increased by incubation with a purified cutaneous collagenase (5). This paper demonstrates the in vivo leukotactic properties of these product peptides released by the activity of this cutaneous collagenase from native soluble collagen. . Materials and Methods-Isotonic saline extracts of rat skin were prepared via the extraction in the cold of 100 g of minced skin with 1000 ml of 0.15 M NaCl in a Lourdes blender for 45 min. After standing in the cold to swell for 16 hr, the supernatant was collected by centrifugation at 12,OOOg for 2 hr. This supernatant was dialyzed exhaustively against water, recen tri f uged, and lyop hilized .This lyophilized skin extract (S1) was activated with 1 mg of crystalline trypsin/lO mg of S1 at pH 5.5 and 22' for 20 min. At this time, 1.1 mg of soybean trypsin inhibitor was added/mg of trypsin; and the solution was made up to 65% with ammonium sulfate in the cold. After centrifuging, the supernatant was dialyzed against water until free of the ammonium ion and lyophilized. The lyophilizate was reconstituted ...
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