Skip to main content

Methods for analysis of matrix metalloproteinase regulation of neutrophil-endothelial cell adhesion

Abstract

Recent evidence indicates novel role for matrix metalloproteinases (MMPs), in particular gelatinase A (MMP-2), in the regulation of vascular biology that are unrelated to their well-known proteolytic breakdown of matrix proteins. We have previously reported that MMP-2 can modulate vascular reactivity by cleavage of the Gly32-Leu33 bound in big endothelin-1 (ET-1) yielding a novel vasoactive peptide ET-1[1–32]. These studies were conducted to investigate whether gelatinolytic MMPs could affect neutrophil-endothelial cell attachment. ET-1[1–32] produced by MMP-2 up-regulated CD11b/CD18 expression on human neutrophils, thereby promoted their adhesion to cultured endothelial cells. ET-1[1–32] evoked release of gelatinase B (MMP-9), which in turn cleaved big ET-1 to yield ET-1[1–32], thus revealing a self-amplifying loop for ET-1[1–32] generation. ET-1[1–32] was rather resistant to cleavage by neutrophil proteases and further metabolism of ET-1[1–32] was not a prerequisite for its biological actions on neutrophils. The neutrophil responses to ET-1[1–32] were mediated via activation of ETA receptors through activation of the Ras/Raf-1/MEK/ERK signaling pathway. These results suggest a novel role for gelatinase A and B in the regulation of neutrophil functions and their interactions with endothelial cells. Here we describe the methods in detail as they relate to our previously published work.

References

  1. 1.

    Nagase H, Woessner JF. Matrix metalloproteinases. J Biol Chem 1999; 274:21491–21494.

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Pruijt JFM et al. Prevention of interleukin-8-induced mobilization of haematopoietic progenitor cells in rhesus monkeys by inhibitory antibodies against gelatinase B (MMP-9). Proc Natl Acad Sci USA 1999; 96:10863–10868.

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    Opdenakker G, Van den Steen PE, Van Damme J. Gelatinase B: a tuner and amplifier of immune functions. Trends Immunol 2001; 22:571–579.

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    Van den Steen PE, Proost P, Wuyts A, Van Damme J, Opdenakker G. Neutrophil gelatinase B potentiates interleukin-8 tenfold by amino terminal processing, whereas it degrades CTAP-III, PF-4, and GRO-α and leaves RANTES and MCP-2 intact. Blood 2000; 96:2673–2681.

    PubMed  Google Scholar 

  5. 5.

    Sawicki G, Salas E, Murat J, Miszta-Lane H, Radomski MW. Release of gelatinase A during platelet activation mediates aggregation. Nature 1997; 386:616–619.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Fernandez-Patron C, Stewart KG, Zhang Y, Koivunen E, Radomski MW, Davidge ST. Matrix metalloproteinase-2-dependent cleavage of calcitonin gene-related peptide promotes vasoconstriction. Circ Res 2000; 87:670–676.

    PubMed  CAS  Google Scholar 

  7. 7.

    Fernandez-Patron C, Radomski MW, Davidge ST. Vascular matrix metalloproteinase-2 cleaves big endothelin-1 yielding a novel vasoconstrictor. Circ Res 1999; 85:906–911.

    PubMed  CAS  Google Scholar 

  8. 8.

    Miyauchi T, Yanagisawa M, Tomizawa T, Sugoshita Y, Suzuki N, Fujino M, Ajisaka R, Goto K, Masaki T. Increased plasma concentration of endothelin-1 and big endothelin-1 in acute myocardial infarction. Lancet 1989; 2:53–54.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Watanabe T, Suzuki N, Shimamoto N, Fujino M, Imada A. Contribution of endogenous endothelin to the extension of myocardial infarct size in rats. Circ Res 1991; 69:370–377.

    PubMed  CAS  Google Scholar 

  10. 10.

    Jenkins GM, Crow MT, Bilato C, Gluzband Y, Ryu WS, Li Z, Stetler-Stevenson W, Nater C, Froehlich JP, Lakatta EG, Cheng L. Increased expression of membrane-type matrix metalloproteinase and preferential localization of matrix metalloproteinase-2 to the neointima of balloon-injured rat carotid arteries. Circulation 1998; 97:82–90.

    PubMed  CAS  Google Scholar 

  11. 11.

    Lerman A, Edwards BS, Hallett JW, Heublein DM, Sandberg SM, Burnett JC Jr. Circulating and tissue endothelin immunoreactivity in advanced atherosclerosis. N Engl J Med 1991; 325:997–1001.

    PubMed  CAS  Google Scholar 

  12. 12.

    Newby AC, Southgate KM, Davies M. Extracellular matrix degrading metalloproteinases in the pathogenesis of arteriosclerosis. Basic Res Cardiol 1993; 89(Suppl. 1):59–70.

    Google Scholar 

  13. 13.

    Lefer AM, Lefer DJ. Pharmacology of the endothelium in ischemia-reperfusion and circulatory shock. Ann Rev Pharmacol Toxicol 1993; 33:71–90.

    Article  CAS  Google Scholar 

  14. 14.

    López-Farré A, Riesco A, Espinosa G, Digiuni E, Cernadas MR, Alvarez V, Monton M, Rivas F, Gallego MJ, Egido J, Casado S, Caramelo C. Effect of endothelin-1 on neutrophil adhesion to endothelial cells and perfused hearts. Circulation 1993; 88:1166–1171.

    PubMed  Google Scholar 

  15. 15.

    Filep JG, Fournier A, Földes-Filep E. Acute proinflammatory actions of endothelin-1 in the guinea pig lung: involvement of ETA and ETB receptors. Br J Pharmacol 1995; 115:227–236.

    PubMed  CAS  Google Scholar 

  16. 16.

    Helset E, Lindal S, Olsen R, Hyklebust R, Jorgensen L. Endothelin-1 causes sequential trapping of platelets and neutrophils in pulmonary microcirculation in rats. Am J Physiol 1996; 271:L538-L546.

    PubMed  CAS  Google Scholar 

  17. 17.

    Zouki C, Baron C, Fournier A, Filep JG. Endothelin-1 enhances neutrophil adhesion to human coronary artery endothelial cells: role of ETA receptors and platelet-activating factor. Br J Pharmacol 1999; 127:969–979.

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    Fernandez-Patron C, Zouki C, Whittal R, Chan JSD, Davidge ST, Filep JG. Matrix metalloproteinases regulate neutrophil-endothelial cell adhesion through generation of ET-1[1–32]. FASEB J 2001; 15:2230–2240.

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    Dai Y, Whittal RM, Li L. Two-layer sample preparation: a method for MALDI-MS analysis of complex peptide and protein mixtures. Anal Chem 1999; 71:1087–1091.

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Filep JG, Delalandre A, Payette Y, Földes-Filep E. The glucocorticoid receptor regulates expression of L-selectin and CD11/Cd18 on human neutrophils. Circulation 1997; 96:295–301.

    PubMed  CAS  Google Scholar 

  21. 21.

    Zouki C, Zhang SL, Chan JSD, Filep JG. Peroxynitrite induces integrin-dependent adhesion of human neutrophils to endothelial cells via activation of the Raf-1/MEK/Erk pathway. FASEB J 2001; 15:25–27 (10.1096/fj.00-0521fje, published online November 9, 2000).

    PubMed  CAS  Google Scholar 

  22. 22.

    Marais R, Wynne J, Treisman R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 1993; 73:381–393.

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Gardner AM, Lange-Carter CA, Vaillancourt RR, Johnson GL. Measuring activation of kinases in mitogen-activated protein kinase regulatory network. Meth Enzymol 1994; 238:258–270.

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    Taylor SJ, Shalloway D. Cell cycle-dependent activation of Ras. Curr Biol 1996; 6:1621–1627.

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    Kaw S, Hecker M, Vane JR. The two step conversion of big endothelin 1 to endothelin 1 by subcellular fractions from human polymorphonuclear leukocytes. Proc Natl Acad Sci USA 1992; 89:6886–6890.

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Zouki C, Beauchamp M, Baron C, Filep JG. Prevention of in vitro neutrophil adhesion to endothelial cells through shedding of L-selectin by C-reactive protein and peptides derived from C-reactive protein. J Clin Invest 1997; 100:522–529.

    PubMed  Article  CAS  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to János G. Filep.

Additional information

Published: October 28, 2002

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Fernandez-Patron, C., Zouki, C., Whittal, R.M. et al. Methods for analysis of matrix metalloproteinase regulation of neutrophil-endothelial cell adhesion. Biol Proced Online 4, 38–48 (2002). https://doi.org/10.1251/bpo32

Download citation

Indexing terms

  • matrix metalloproteinases
  • big endothelin-1
  • endothelin-1(1-32)
  • neutrophil granulocytes
  • endothelial cells
  • adhesion molecules
  • MAPK signaling
  • leukocyte trafficking
  • inflammation
  • innate immunity