Extracellular collagenase, proteoglycanase and products of their activity, released in organ culture by intact dermal inflammatory lesions produced by sulfur mustard

Peak (1 and 2 d) and healing (3, 6, and 10 d) inflammatory lesions were produced in rabbits by the topical application of the military vesicant, bis(2-chloroethyl)sulfide, commonly called sulfur mustard (SM). SM produces an acute sterile dermal inflammatory reaction with little or no necrosis, except in the epidermis, which dies during the first day. After an animal was killed, its lesions were excised intact, as full-thickness 1.0-cm2 explants. They were then organ-cultured for 3 d in order to maintain the viability of both local and infiltrating cells. The extracellular fluid in each lesion equilibrated with the culture fluid, which was collected daily and analyzed for collagenase and proteoglycanase activities. These metalloproteinase activities were measured after we had i) destroyed the alpha-macroglobulin inhibitors with KSCN, ii) destroyed the tissue inhibitor of metalloproteinases (TIMP) by reduction and alkylation, and iii) activated the latent proteinase activity with aminophenylmercuric acetate (APMA). Hydroxyproline-containing peptides and glycosaminoglycans (GAG) released into the culture fluids were also measured as indicators of local collagenase and proteoglycanase activity within the inflammatory lesions. In general, the levels of both the metalloproteinases and the products of their activity were higher in second- and third-day culture fluids than in first-day culture fluids, and higher in fluids from SM lesions than in those from normal skin. The activated fibroblast was apparently the major cell type producing the collagenase and proteoglycanase. The hydrolysis of collagen and ground substance occurs pericellularly. An excess of inhibitors exists outside the pericellular region. The daily change in culture fluids apparently decreased such inhibitors, so that by the second and third day of culture we could detect the changes in pericellular enzyme activity that were not detectable on the first day of culture. As the inflammatory lesions healed, the extracellular enzyme products (hydroxyproline and GAG) increased more than the enzymes that produced these products. With healing, a decrease occurs in the extravasation of all serum components, especially the large ones such as the alpha-macroglobulin inhibitors. We propose that during healing, the decrease in these inhibitors allows the metalloproteinases to begin the remodeling process, and that during the peak phase of inflammation, these same inhibitors protect extracellular matrix against hydrolysis by such proteinases.