Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1982 Oct;70(4):845–852. doi: 10.1172/JCI110681

Proteolysis by Neutrophils

RELATIVE IMPORTANCE OF CELL-SUBSTRATE CONTACT AND OXIDATIVE INACTIVATION OF PROTEINASE INHIBITORS IN VITRO

Edward J Campbell 1, Robert M Senior 1, John A McDonald 1, David L Cox 1, Jeanne M Greco 1, Jill A Landis 1
PMCID: PMC370293  PMID: 6181097

Abstract

Polymorphonuclear leukocytes have been implicated in connective tissue injury in a variety of disease processes. To gain insight into mechanisms by which neutrophils might degrade connective tissue macromolecules in the presence of proteinase inhibitors, we have used a model system that allows neutrophils to be held in vitro under physiologic conditions in close proximity to a very proteinase-sensitive substrate, 125I-labeled fibronectin. We have found: (a) neutrophils spread rapidly on the fibronectin substrate; (b) fibronectin proteolysis by neutrophils is largely attributable to released elastase, and is linearly related to cell number over the range of 2,000 to 30,000 cells per assay; (c) oxidants released from neutrophils stimulated by opsonized zymosan or phorbol myristate acetate do not protect released elastase from inhibition by α1-proteinase inhibitor or α2-macroglobulin; (d) neutrophil myeloperoxidase and enzymatically generated superoxide anion render α1-proteinase inhibitor ineffective against fibronectin proteolysis when neutrophils are added 30 min later; and (e) α1-proteinase inhibitor and α2-macroglobulin incompletely inhibit fibronectin proteolysis by neutrophils (79.8±6.3 and 73.5±12.0%, respectively.) The data suggested that proteolysis due to neutrophils that are in contact with susceptible macromolecules may occur due to partial exclusion of inhibitors from the cell-substrate interface. Although confirming that α1-proteinase inhibitor is ineffective against neutrophil-derived proteolysis after exposure to oxidants, these studies did not support the hypothesis that oxidants released from stimulated neutrophils enhance activity of proteinases they release in the presence of α1-proteinase inhibitor. We anticipate that further studies with this test system will be helpful in defining conditions that modulate inflammatory connective tissue injury in diseases such as pulmonary emphysema and rheumatoid arthritis.

Full text

PDF
845

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Babior B. M., Kipnes R. S., Curnutte J. T. Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest. 1973 Mar;52(3):741–744. doi: 10.1172/JCI107236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Babul J., Stellwagen E. Measurement of protein concentration with interferences optics. Anal Biochem. 1969 Apr 4;28(1):216–221. doi: 10.1016/0003-2697(69)90172-9. [DOI] [PubMed] [Google Scholar]
  3. Brot N., Weissbach L., Werth J., Weissbach H. Enzymatic reduction of protein-bound methionine sulfoxide. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2155–2158. doi: 10.1073/pnas.78.4.2155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl. 1968;97:77–89. [PubMed] [Google Scholar]
  5. Campbell E. J., White R. R., Senior R. M., Rodriguez R. J., Kuhn C. Receptor-mediated binding and internalization of leukocyte elastase by alveolar macrophages in vitro. J Clin Invest. 1979 Sep;64(3):824–833. doi: 10.1172/JCI109530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carp H., Janoff A. In vitro suppression of serum elastase-inhibitory capacity by reactive oxygen species generated by phagocytosing polymorphonuclear leukocytes. J Clin Invest. 1979 Apr;63(4):793–797. doi: 10.1172/JCI109364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carp H., Janoff A. Possible mechanisms of emphysema in smokers. In vitro suppression of serum elastase-inhibitory capacity by fresh cigarette smoke and its prevention by antioxidants. Am Rev Respir Dis. 1978 Sep;118(3):617–621. doi: 10.1164/arrd.1978.118.3.617. [DOI] [PubMed] [Google Scholar]
  8. Carp H., Janoff A. Potential mediator of inflammation. Phagocyte-derived oxidants suppress the elastase-inhibitory capacity of alpha 1-proteinase inhibitor in vitro. J Clin Invest. 1980 Nov;66(5):987–995. doi: 10.1172/JCI109968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Castillo M. J., Nakajima K., Zimmerman M., Powers J. C. Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases. Anal Biochem. 1979 Oct 15;99(1):53–64. doi: 10.1016/0003-2697(79)90043-5. [DOI] [PubMed] [Google Scholar]
  10. Clark R. A., Stone P. J., El Hag A., Calore J. D., Franzblau C. Myeloperoxidase-catalyzed inactivation of alpha 1-protease inhibitor by human neutrophils. J Biol Chem. 1981 Apr 10;256(7):3348–3353. [PubMed] [Google Scholar]
  11. Cohen A. B. The effects in vivo and in vitro of oxidative damage to purified alpha1-antitrypsin and to the enzyme-inhibiting activity of plasma. Am Rev Respir Dis. 1979 Jun;119(6):953–960. doi: 10.1164/arrd.1979.119.6.953. [DOI] [PubMed] [Google Scholar]
  12. Gadek J. E., Fells G. A., Crystal R. G. Cigarette smoking induces functional antiprotease deficiency in the lower respiratory tract of humans. Science. 1979 Dec 14;206(4424):1315–1316. doi: 10.1126/science.316188. [DOI] [PubMed] [Google Scholar]
  13. Gadek J. E., Fells G. A., Wright D. G., Crystal R. G. Human neutrophil elastase functions as a type III collagen "collagenase". Biochem Biophys Res Commun. 1980 Aug 29;95(4):1815–1822. doi: 10.1016/s0006-291x(80)80110-0. [DOI] [PubMed] [Google Scholar]
  14. Janoff A. At least three human neutrophil lysosomal proteases are capable of degrading joint connective tissues. Ann N Y Acad Sci. 1975 Jun 13;256:402–408. doi: 10.1111/j.1749-6632.1975.tb36066.x. [DOI] [PubMed] [Google Scholar]
  15. Janoff A., Carp H., Lee D. K., Drew R. T. Cigarette smoke inhalation decreases alpha 1-antitrypsin activity in rat lung. Science. 1979 Dec 14;206(4424):1313–1314. doi: 10.1126/science.316187. [DOI] [PubMed] [Google Scholar]
  16. Janoff A., Carp H. Possible mechanisms of emphysema in smokers: cigarette smoke condensate suppresses protease inhibition in vitro. Am Rev Respir Dis. 1977 Jul;116(1):65–72. doi: 10.1164/arrd.1977.116.1.65. [DOI] [PubMed] [Google Scholar]
  17. Janoff A., Sloan B., Weinbaum G., Damiano V., Sandhaus R. A., Elias J., Kimbel P. Experimental emphysema induced with purified human neutrophil elastase: tissue localization of the instilled protease. Am Rev Respir Dis. 1977 Mar;115(3):461–478. doi: 10.1164/arrd.1977.115.3.461. [DOI] [PubMed] [Google Scholar]
  18. Johnson D. A. Ozone inactivation of human alpha 1-proteinase inhibitor. Am Rev Respir Dis. 1980 Jun;121(6):1031–1038. doi: 10.1164/arrd.1980.121.6.1031. [DOI] [PubMed] [Google Scholar]
  19. Johnson D., Travis J. Structural evidence for methionine at the reactive site of human alpha-1-proteinase inhibitor. J Biol Chem. 1978 Oct 25;253(20):7142–7144. [PubMed] [Google Scholar]
  20. Johnson K. J., Varani J. Substrate hydrolysis by immune complex-activated neutrophils: effect of physical presentation of complexes and protease inhibitors. J Immunol. 1981 Nov;127(5):1875–1879. [PubMed] [Google Scholar]
  21. Mainardi C. L., Dixit S. N., Kang A. H. Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules. J Biol Chem. 1980 Jun 10;255(11):5435–5441. [PubMed] [Google Scholar]
  22. Martodam R. R., Baugh R. J., Twumasi D. Y., Liener I. E. A rapid procedure for the large scale purification of elastase and cathepsin G from human sputum. Prep Biochem. 1979;9(1):15–31. doi: 10.1080/00327487908061669. [DOI] [PubMed] [Google Scholar]
  23. Matheson N. R., Wong P. S., Schuyler M., Travis J. Interaction of human alpha-1-proteinase inhibitor with neutrophil myeloperoxidase. Biochemistry. 1981 Jan 20;20(2):331–336. doi: 10.1021/bi00505a016. [DOI] [PubMed] [Google Scholar]
  24. Matheson N. R., Wong P. S., Travis J. Enzymatic inactivation of human alpha-1-proteinase inhibitor by neutrophil myeloperoxidase. Biochem Biophys Res Commun. 1979 May 28;88(2):402–409. doi: 10.1016/0006-291x(79)92062-x. [DOI] [PubMed] [Google Scholar]
  25. Matheson N. R., Wong P. S., Travis J. Isolation and properties of human neutrophil myeloperoxidase. Biochemistry. 1981 Jan 20;20(2):325–330. doi: 10.1021/bi00505a015. [DOI] [PubMed] [Google Scholar]
  26. McCord J. M., Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem. 1969 Nov 25;244(22):6049–6055. [PubMed] [Google Scholar]
  27. McDonald J. A., Baum B. J., Rosenberg D. M., Kelman J. A., Brin S. C., Crystal R. G. Destruction of a major extracellular adhesive glycoprotein (fibronectin) of human fibroblasts by neutral proteases from polymorphonuclear leukocyte granules. Lab Invest. 1979 Mar;40(3):350–357. [PubMed] [Google Scholar]
  28. McDonald J. A., Kelley D. G. Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments. J Biol Chem. 1980 Sep 25;255(18):8848–8858. [PubMed] [Google Scholar]
  29. Mosesson M. W., Umfleet R. A. The cold-insoluble globulin of human plasma. I. Purification, primary characterization, and relationship to fibrinogen and other cold-insoluble fraction components. J Biol Chem. 1970 Nov 10;245(21):5728–5736. [PubMed] [Google Scholar]
  30. Osserman E. F., Lawlor D. P. Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia. J Exp Med. 1966 Nov 1;124(5):921–952. doi: 10.1084/jem.124.5.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Roughley P. J. The degradation of proteoglycan by leucocyte elastase. Biochem Soc Trans. 1977;5(2):443–445. doi: 10.1042/bst0050443. [DOI] [PubMed] [Google Scholar]
  32. Senior R. M., Tegner H., Kuhn C., Ohlsson K., Starcher B. C., Pierce J. A. The induction of pulmonary emphysema with human leukocyte elastase. Am Rev Respir Dis. 1977 Sep;116(3):469–475. doi: 10.1164/arrd.1977.116.3.469. [DOI] [PubMed] [Google Scholar]
  33. Varani J., Johnson K., Kaplan J. Development of a solid-phase assay for measurement of proteolytic enzyme activity. Anal Biochem. 1980 Sep 15;107(2):377–384. doi: 10.1016/0003-2697(80)90399-1. [DOI] [PubMed] [Google Scholar]
  34. Weiss S. J., Rustagi P. K., LoBuglio A. F. Human granulocyte generation of hydroxyl radical. J Exp Med. 1978 Feb 1;147(2):316–323. doi: 10.1084/jem.147.2.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wong P. S., Travis J. Isolation and properties of oxidized alpha-1-proteinase inhibitor from human rheumatoid synovial fluid. Biochem Biophys Res Commun. 1980 Oct 16;96(3):1449–1454. doi: 10.1016/0006-291x(80)90113-8. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES