Chemotactic factors released in culture by intact developing and healing skin lesions produced in rabbits by the irritant sulfur mustard.

Development, peak and healing lesions were induced in the skin of rabbits by topical applications (on different days) of the chemical irritant sulfur mustard (SM). Immediately after the rabbits were euthanized, the intact lesions were excised and organ-cultured for 17 to 20 hours. The culture fluids from early, peak and healing SM lesions all showed high chemotactic activity for both PMN and MN. This finding suggests that the PMN and MN, seen microscopically in tissue sections of the lesions, were entering continuously, even during the healing process. The chemotaxins identified were the eicosanoid LTB4, the chemokine IL-8, and proteases producing the complement fragment C5a. Other studies from our laboratory showed that the number of cells containing IL-1, IL-8, MCP-1, and GRO mRNAs was increased in SM lesions. Chemotactic activity was released by both live and dead (frozen and thawed) cell suspensions of PMN, MN, and fibroblasts, suggesting that these cells were major sources of the chemotaxins produced by the SM lesion explants. Explants of normal skin produced considerable chemotactic activity for MN, but not for PMN. Chemotactic activity for PMN, and the release of LTB4, IL-8 and proteases cleaving C5 to C5a, occurred only in explants infiltrated by leukocytes.

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.

Full-thickness human skin explants for testing the toxicity of topically applied chemicals.

This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36 degrees C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components. The toxicant type seemed to be caused by an invagination of the plasma membrane. Only toxicant-type vacuoles increased appreciably in number when skin explants were exposed to mustard, and to other toxicants.

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.

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-amide(Boc-Leu -Gly- Arg-AFC, herein abbreviated LGA-AFC), and (2) N-benzoyl-phenylalanine-beta-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 with alpha-macroglobulin proteinase inhibitors (alpha M). Endopeptidases, but not exopeptidases, are entrapped and inhibited by alpha M, because an internal peptide band in alpha M must first be hydrolyzed before molecular rearrangement (required for proteinase inhibition) occurs. The catalytic site of endopeptidases that are entrapped and inhibited by alpha M is known to remain active on (and reachable by) small synthetic peptide substrates such as LGA-AFC and BPN.(ABSTRACT TRUNCATED AT 400 WORDS)

Sources of extracellular lysosomal enzymes released in organ-culture by developing and healing inflammatory lesions.

Developing and healing inflammatory lesions were topically produced in the skin of rabbits by sulfur mustard (SM). After the rabbits were sacrificed, the various lesions were removed and organ-cultured. The organ-culture fluids extracted the extracellular lysosomal enzymes (acid phosphatase, beta-glucuronidase, beta-galactosidase, and lysozyme), so that they could be measured biochemically along with lactic dehydrogenase (LDH), an enzyme marker for cell death. In tissue sections, the number and types of cells were counted, and their lysosomal enzyme content evaluated histochemically. The culture fluids from peak lesions contained much lower levels of all five enzymes than did culture fluids from healing lesions. When histological-histochemical-biochemical correlations were made, serum, macrophages (MN), and activated fibroblasts (but not tissue PMN) appeared to be major sources of extracellular lysosomal enzymes in peak lesions; and the dead PMN in the crusts and the activated fibroblasts in the tissues appeared to be major sources in healing lesions. The high lysosomal enzyme content of the crusts covering the lesions suggests that this passive barrier may also play an active role in promoting healing and in protecting against invasion by microorganisms.

Inflammatory mediators and modulators released in organ culture from rabbit skin lesions produced in vivo by sulfur mustard. III. Electrophoretic protein fractions, trypsin-inhibitory capacity, alpha 1-proteinase inhibitor, and alpha 1- and alpha 2-macroglobulin proteinase inhibitors of culture fluids and serum.

This is the third report in a series on the inflammatory mediators and modulators released in organ culture from skin lesions of various ages, which were produced in vivo in rabbits by the military vesicant, sulfur mustard (SM). It describes the electrophoretic protein fractions and trypsin-inhibitory capacities of the various culture fluids and the amounts of alpha 1-proteinase inhibitor and alpha-macroglobulin proteinase inhibitors in these fluids. With one-dimensional electrophoresis, the albumin and beta-globulin fractions of protein in culture fluids varied little with the development and healing of the SM lesions. These fractions proportionally resembled the corresponding fractions found in serum. The alpha 1-globulin fraction was proportionally smaller than the corresponding fractions of serum as the lesions healed. The alpha 2-globulin fraction was proportionally smaller than the corresponding fractions of serum at all stages of lesion development and healing. The gamma-globulin fraction was proportionally larger as the lesions healed. With two-dimensional electrophoresis, about 68%, 46%, and 35% of the protein spots in culture fluids from representative 1-day and 6-day SM lesions and normal skin, respectively, matched those from serum. In each case, the large, diffuse, serum albumin spot represented about two-thirds of the protein present. Thus, gravimetrically, in normal skin and in both developing and healing lesions, the extracellular proteins were 80-90% of serum origin. The trypsin-inhibitory capacity (TIC) per milligram protein in the culture fluids of healing lesions was markedly less than the TIC per milligram protein in the fluids of peak lesions. This decrease correlates well with the decrease found in the alpha 1-globulin fraction, which contains alpha 1-antiproteinase (alpha 1-PI) (and alpha 1-macroglobulin [alpha 1M] in rabbits). The alpha 1PI and the alpha 1M-alpha 2M proteinase inhibitors were identified in the culture fluids by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blots, specific antibodies, and the immuno-peroxidase technique. The levels of both free and proteinase-complexed alpha 1PI and alpha M inhibitors in the culture fluids decreased as the lesions healed. In both developing and healing lesions, at least half of the alpha 1PI and alpha M inhibitors seemed to be complexed with proteinases. Thus, serum seems to be a major source of unbounded extracellular protein within acute inflammatory lesions, and serum proteinase inhibitors seem to be the host's major defense against local damage by proteinases from serum, infiltrating leukocytes, and activated fibroblasts.

Inflammatory mediators and modulators released in organ culture from rabbit skin lesions produced in vivo by sulfur mustard. I. Quantitative histopathology; PMN, basophil, and mononuclear cell survival; and unbound (serum) protein content.

When applied topically to the skin of rabbits in vivo, sulfur mustard (SM), the vesicant used in World War I, produced a slowly developing inflammatory response, which peaked in size at 1 and 2 days, ulcerated within 3 days, and reepithelialized by 10 days. Histologically, basophils and polymorphonuclear leukocytes (PMNs) were common in both early and late lesions, and the crust over the ulcers was composed of dead epidermal cells, fibrin, and large numbers of PMNs. Healing occurred under the crust by migration of epidermal cells from the margins of the lesions and from the hair follicles. In organ culture, the lesion explants survived well, and reepithelialization even took place. Their excellent survival enabled us to compare the life spans of the infiltrating leukocytes within an inflammatory site. PMNs within the explants began disappearing during the first day of culture, and almost all had disappeared by 3 days. In contrast, over half of the basophils and the mononuclear cells within the explants were still present after 3 days of culture. The 1-, 2-, 3-, 6-, and 10-day (1.0-sq cm) SM lesion biopsies showed a 30-45% increase in weight (when compared with normal skin), presumably due to the extravasation of serum proteins and the fluids retained by them. When the biopsies were organ-cultured for 3 days, the 1-, 2-, and 3-day lesions lost weight, and the 6- and 10-day lesions (and normal skin) gained weight. These weight differences were not due to the amount of unbound protein extractable into the culture fluids, because both the early lesions and the late lesions contained about the same amount of unbound protein. The most likely explanation for these weight differences is that the newly formed ground substances of late lesions absorbed culture fluid, because the ground substance had changed from the sol state of acute inflammation (in which it was extractable) back to its normal gel state (in which it was not extractable). The unbound protein extractable into the culture fluids was mostly of serum origin. This protein averaged 1.9 mg for 1.0 sq cm normal skin explants (with a mean weight of 215 mg), and 6.4 mg for 1-day SM lesions (with a mean weight of 313 mg). Because rabbit serum contains about 60 mg protein/ml, these figures indicate that normal skin contained about 15% (unbound) serum by weight.(ABSTRACT TRUNCATED AT 400 WORDS)

Inflammatory mediators and modulators release in organ culture from rabbit skin lesions produced in vivo by sulfur mustard. II. Evans blue dye experiments that determined the rates of entry and turnover of serum protein in developing and healing lesions.

Extravasated serum seems to be the major modulator of the local inflammatory response, because it provides both proinflammatory and antiinflammatory components. This report describes the rates of entry and turnover of extravasated serum protein in dermal inflammatory lesions produced by the military vesicant sulfur mustard (SM). Rabbits, bearing SM skin lesions, were given an intravenous injection of Evans blue dye, so that at the time of sacrifice, 2 hours later, their skin lesions were 2 hours and 1,2,3,6, and 10 days of age. Evans blue labels serum albumin, a representative serum protein. By multiplying the amount of Evans blue contained in the lesions by a factor that converted micrograms of Evans blue into milligrams of serum protein, the authors could estimate the 2-hour rate of entry of serum protein into these lesions. Serum protein in the lesions was both bound and unbound. The unbound protein was extractable from the lesions into the culture fluids, and, electrophoretically, was similar in composition to serum protein. Grossly edematous peak lesions (1 day of age) contained 7.8 mg of unbound serum protein per square centimeter of skin. Healing lesions (6 and 10 days of age) contained about 4.5 mg/sq cm, and normal skin about 1.7 mg/sq cm. Lesions 1 day of age had the highest rate of serum albumin entry, and about 36% of this Evans-blue-labeled protein was unbound, ie, extractable into the culture fluids. Lesions 3 and 6 days of age had a rate of serum albumin entry that was roughly half that of 1-day lesions, and only about 13% of this entering protein was unbound. Normal skin had a very low rate of serum albumin entry, and only 8% of this entering protein was unbound. The turnover rate of the unbound (extractable) serum protein could be estimated from the 2-hour entry rate of the Evans-blue-labeled albumin and the total protein in the culture fluids. In 1-day lesions, about 25% of the serum protein in the culture fluids was protein which had entered during the last 2 hours, so that 100% of this unbound protein should have been replaced once in 8 hours. In contrast, in 3- and 6-day lesions, this unbound serum protein should have been replaced once in about 35 hours, and in normal skin once in 80 hours. Evans-blue-labeled serum albumin continuously entered both the bound and unbound compartments of the SM lesions, even during the healing stages.(ABSTRACT TRUNCATED AT 400 WORDS)