Inflammation

, Volume 12, Issue 4, pp 311–334 | Cite as

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

  • Kazuyuki Higuchi
  • Akira Kajiki
  • Masahiro Nakamura
  • Susumu Harada
  • Peggy J. Pula
  • Alan L. Scott
  • Arthur M. DannenbergJr.
Original Articles

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.

Keywords

Plasmin Chymotrypsin Inflammatory Lesion Endopeptidase Culture Fluid 

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References

  1. 1.
    Robinson, W. A., andA. Magalik. 1975. The kinetics and regulation of granulopoiesis.In Neutrophil Physiology and Pathology. J. R. Humbert, P. A. Miescher, and E. R. Jaffe, editors. Grune & Stratton, New York.Google Scholar
  2. 2.
    Weissmann, G. (ed.). 1980. The Cell Biology of Inflammation. Handbook of Inflammation, Vol. 2, L. E. Glynn, J. C. Houck and G. Weissmann, editors. Elsevier/North-Holland, Amsterdam.Google Scholar
  3. 3.
    Oppenheim, J. J., D. L. Rosenstreich, andM. Potter. 1981. Cellular Function in Immunity and Inflammation. Elsevier/North-Holland, New York.Google Scholar
  4. 4.
    Movat, H. Z. 1985. The Inflammatory Reaction. Elsevier, Amsterdam.Google Scholar
  5. 5.
    Dannenberg, A. M., Jr., P. J. Pula, L. H. Liu, S. Harada, F. Tanaka, R. F. Vogt, Jr., A. Kajiki, K. Higuchi. 1985. 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.Am. J. Pathol. 121:15–27.Google Scholar
  6. 6.
    Vogt, R. F., Jr, A. M. Dannenberg, Jr., B. H. Schofield, N. A. Hynes, andB. Papir-Meister. 1984. Pathogenesis of skin lesions caused by sulfur mustard.Fundam. Appl. Toxicol. 4:S71-S83.Google Scholar
  7. 7.
    Papirmeister, B., C. L. Gross, J. P. Petrali, andC. J. Hixson. 1984. Pathology produced by sulfur mustard in human skin grafts on athymic nude mice. I. Gross and light microscopic changes.J. Toxicol. Cutic. Ocular Toxicol. 3:371–391.Google Scholar
  8. 8.
    Papirmeister, B., C. L. Gross, J. P. Petrali, andC. J. Hixson. 1984. Pathology produced by sulfur mustard in human skin grafts on athymic nude mice. II. Ultrastructural changes.J. Toxicol. Cutic. Ocular Toxicol. 3:393–408.Google Scholar
  9. 9.
    Harada, S., A. M. Dannenberg, Jr., R. F. Vogt, Jr., J. E. Myrick, F. Tanaka, L. C. Redding, R. M. Merkhofer, P. J. Pula, andA. L. Scott. 1987. 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, andα 1proteinase inhibitor, andα 1 andα 2-macroglobulin proteinase inhibitors of culture fluids and serum.Am. J. Pathol. 126:148–163.Google Scholar
  10. 10.
    Kajiki, A., K. Higuchi, M. Nakamura, L. H. Liu, P. J. Pula, andA. M. Dannenberg, Jr. 1988. Sources of extracellular lysosomal enzymes released in organ culture by developing and healing inflammatory lesions.J. Leukocyte Biol. 43:104–116.Google Scholar
  11. 11.
    Huseby, R. M., S. A. Clavin, R. E. Smith, R. N. Hull, andE. L. Smithwick, Jr. 1977. Studies on tissue culture plasminogen activator. II. The detection and assay of urokinase and plasminogen activator from LLC-PK1 cultures (porcine) by the synthetic substrate N-benz-yloxycarbonyl-glycyl-glycyl-arginyl-4-methoxy-2-naphthylamide.Thromb. Res. 10:679–687.Google Scholar
  12. 12.
    Nieuwenhuizen, W., G. Wijngaards, andE. Groeneveld. 1977. Fluorogenic peptide amide substrates for the estimation of plasminogen activators and plasmin.Anal. Biochem. 83:143–148.Google Scholar
  13. 13.
    Smith, R. E., E. R. Bissell, A. R. Mitchell, andK. W. Pearson. 1980. Direct photometric or fluorometric assay of proteinases using substrates containing 7-amino-4-trifluoromethyl-coumarin.Thromb. Res. 17:393–402.Google Scholar
  14. 14.
    Dannenberg, A. M., Jr., andW. E. Bennett. 1964. Hydrolytic enzymes of rabbit mono-nuclear exudate cells. I. Quantitative assay and properties of their proteases, nonspecific esterases and lipase.J. Cell Biol. 21:1–13.Google Scholar
  15. 15.
    Rojas-Espinosa, O., P. Arce-Paredez, A. M. Dannenberg, Jr., andR. L. Kamenetz. 1975. Macrophage esterase: Identification, purification and properties of chymotrypsin-like esterase from lung that hydrolyses and transfers nonpolar amino acid esters.Biochim. Biophys. Acta 403:161–179.Google Scholar
  16. 16.
    Castillo, M. J., K. Nakajima, M. Zimmerman, andJ. C. Powers. 1979. Sensitive substrates for human leukocyte and porcine pancreatic elastase: A study of the merits of various chromophoric and fluorogenic cleaving groups in assays for serine proteases.Anal. Biochem. 99:53–64.Google Scholar
  17. 17.
    Bradford, M. M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72:248–254.Google Scholar
  18. 18.
    Harada, S., A. M. Dannenberg, Jr., A. Kajiki, K. Higuchi, F. Tanaka, andP. J. Pula. 1985. Inflammatory mediators and modulators released in organ culture from rabbit skin lesions produced in vivo by sulfur mustard: II. Evans blue dye experiments which determined the rates of entry and turnover of serum proteins in developing and healing lesions.Am. J. Pathol. 121:28–38.Google Scholar
  19. 19.
    Dano, K., andE. Reich. 1979. Plasminogen activator from cells transformed by oncogenic virus.Biochim. Biophys. Acta 566:138–151.Google Scholar
  20. 20.
    Schumacher, G. F. B., andW-B. Schill. 1972. Radial diffusion in gel for microdetermination of enzymes. II. Plasminogen activator, elastase, and nonspecific proteases.Anal. Biochem. 48:9–26.Google Scholar
  21. 21.
    Brakman, P., andT. Astrup. 1971. The fibrin plate method for assay of fibrinolytic agents.In Thrombosis and Bleeding Disorder. N. U. Bang, F. K. Beller, E. Deutsch, and E. F. Mammen, editors. Academic Press, New York. 332.Google Scholar
  22. 22.
    Hashimoto, K., K. M. Shafran, P. S. Webber, G. S. Lazarus, andK. H. Singer. 1983. Anti-cell surface pemphigus autoantibody stimulates plasminogen activator activity of human epidermal cells. A mechanism for the loss of epidermal cohesion and blister formation.J. Exp. Med. 157:259–272.Google Scholar
  23. 23.
    Iwanaga, S., T. Morita, H. Kato, T. Harada, N. Adachi, T. Sugo, I. Maruyama, K. Takada, T. Kimura, andS. Sakakibara. 1979. Fluorogenic peptide substrates for proteases in blood coagulation, kallikrein-kinin and fibrinolysis systems.In Kinins-II. Biochemistry; Pathophysiology and Clinical Aspects. S. Fujii, T. Suzuki, and H. Moriya, editors. Plenum Publishing, New York. 147–163.Google Scholar
  24. 24.
    Caporale, L. H., S.-S. Gaber, W. Kell, andO. Götze. 1981. A fluorescent assay for complement activation.J. Immunol. 126:1963–1965.Google Scholar
  25. 25.
    Starkey, P. M., andA. J. Barrett. 1977.α 2-Macroglobulm, a physiological regulator of proteinase activity.In Proteinases in Mammalian Cells and Tissues. A. J. Barrett, editor. Elsevier/North Holland Biomedical Press, Amsterdam. 663–696.Google Scholar
  26. 26.
    Kaplan, A. P. 1981. Coagulation, kinins, and inflammation.In Cellular Functions in Immunity and Inflammation. J. J. Oppenheim, D. L. Rosenstreich and M. Potter, editor. Eisevier/ North Holland, New York, 397–410.Google Scholar
  27. 27.
    Harris, E. D., Jr., andE. C. Cartwright. 1977. Mammalian collagenases.In Proteinases in Mammalian Cells and Tissues. A. J. Barrett, editor. Elsevier/North Holland Biomedical Press, Amsterdam. 249–283.Google Scholar
  28. 28.
    Burleigh, M. C. 1977. Degradation of collagen by non-specific proteinases.In Proteinases in Mammalian Cells and Tissues. A. J. Barrett, editor. Elsevier/North Holland Biomedical Press, Amsterdam. 285–309.Google Scholar
  29. 29.
    Glynn, L. E. (ed.). 1981. Tissue Repair and Regeneration. Handbook of Inflammation, Vol. 3. G. E. Glynn, J. C. Houck, and G. Weissmann, editors. Elsevier/North-Holland Biomedical Press, Amsterdam.Google Scholar
  30. 30.
    Rojas-Espinosa, O., A. M. Dannenberg, Jr., L. A. Sternberger, andT. Tsuda. 1974. The role of cathepsin D in the pathogenesis of tuberculosis: A histochemical study employing unlabeled antibodies and the peroxidase-antiperoxidase complex.Am. J. Pathol. 74:1–18.Google Scholar
  31. 31.
    Suga, M., A. M. Dannenberg, Jr., andS. Higuchi. 1980. Macrophage functional hetero-geneity in vivo: Macrolocal and microlocal macrophage activation, identified by double-staining tissue sections of BCG granulomas for pairs of enzymes.Am. J. Pathol. 99:305–324.Google Scholar
  32. 32.
    Starkey, P. M. 1977. Elastase and cathepsin G; the serine proteinases of human neutrophil leucocytes and spleen.In Proteinases in Mammalian Cells and Tissues. A. J. Barrett, editor. Elsevier/North Holland Biomedical Press, Amsterdam. 57–89.Google Scholar
  33. 33.
    Lagunoff, D., andP. Pritzl. 1976. Characterization of mast cell granule proteins.Arch. Biochem. Biophys. 173:554–563.Google Scholar
  34. 34.
    Schechter, N. M., J. E. Fraki, J. C. Geesin, andG. S. Lazarus. 1983. Human skin chymotryptic proteinase: Isolation and relation to cathepsin G and rat mast cell proteinase I.J. Biol. Chem. 258:2973–2978.Google Scholar
  35. 35.
    Collen, D., andB. Wiman. 1979. Introduction to the round table conference.In The Physiological Inhibitors of Blood Coagulation and Fibrinolysis. D. Collen, B. Wiman, and M. Verstraete, editor. Elsevier/North-Holland Biomedical Press, Amsterdam. 3–4.Google Scholar
  36. 36.
    Harpel, P. C. 1981.α 2-Plasmin inhibitor andα 2-macroglobulin-plasmin complexes in plasma: Quantitation by an enzyme-linked differential antibody immunosorbent assay.J. Clin. Invest. 68:46–55.Google Scholar
  37. 37.
    Tanaka, F., K.Higuchi, M.Nakamura, P. J.Pula, T. E.Hugli, K. G.Moore, R. G.Discipio, J. L.Wagner, G. S.Habicht, G.Beck, D. S.Newcombe, and A. M.Dannen-Berg, Jr. Chemotaxis of granulocytes and macrophages by organ culture fluids from developing and healing dermal sulfur mustard lesions: Role of complement components, leukotriene B4, and interleukin I (in preparation).Google Scholar
  38. 38.
    Woessner, J. F., Jr., K.Higuchi, A.Kajiki, P. J.Pula, and A. M.Dannenberg, Jr. Proteoglycanase and collagenase released in organ culture by acute dermal inflammatory lesions produced by sulfur mustard (in preparation).Google Scholar
  39. 39.
    Pula, P. J., C. L.Ruppert, A. M.Dannenberg, Jr., A.Kajiki, K.Higuchi, N. M.Dahms, J. S.Kerr, and G. W.Hart. Hexosamine-containing and hydroxyproline-containing extracellular matrix components released in organ culture by acute dermal inflammatory lesions produced by sulfur mustard (in preparation).Google Scholar
  40. 40.
    Tanaka, T., B. J. McRae, K. Cho, R. Cook, J. E. Fraki, D. A. Johnson, andJ. C. Pow-Ers. 1983. Mammalian tissue trypsin-like enzymes: Comparative reactivities of human skin tryptase, human lung tryptase, and bovine trypsin with peptide 4-nitroanilide and thioester substrates.J. Biol. Chem. 258:13552–13557.Google Scholar
  41. 41.
    Powers, J. C., T. Tanaka, J. W. Harper, Y. Minematsu, L. Barker, D. Lincoln, K. V. Crumley, J. E. Fraki, N. M. Schechter, G. S. Lazarus, K. Nakajima, K. Nakashino, H. Neurath, andR. G. Woodbury. 1985. Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors.Biochemistry.24:2048–2058.Google Scholar

Copyright information

© Plenum Publishing Corporation 1988

Authors and Affiliations

  • Kazuyuki Higuchi
    • 1
    • 3
  • Akira Kajiki
    • 1
    • 3
  • Masahiro Nakamura
    • 1
    • 3
  • Susumu Harada
    • 1
    • 3
  • Peggy J. Pula
    • 1
    • 3
  • Alan L. Scott
    • 2
    • 3
  • Arthur M. DannenbergJr.
    • 1
    • 3
    • 4
  1. 1.Departments of Environmental Health SciencesThe Johns Hopkins UniversityBaltimore
  2. 2.Departments of Immunology and Infectious DiseasesThe Johns Hopkins UniversityBaltimore
  3. 3.Departments of Epidemiology School of Hygiene and Public HealthThe Johns Hopkins UniversityBaltimore
  4. 4.Department of Pathology, School of MedicineThe Johns Hopkins UniversityBaltimore

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