Skip to main content
NCBI home page
Immunology logoLink to Immunology
. 1991 Sep;74(1):114–120.

Fibronectin fragments released from phorbol ester-stimulated pulmonary artery endothelial cell monolayers promote neutrophil chemotaxis.

L E Odekon 1, M B Frewin 1, P Del Vecchio 1, T M Saba 1, P W Gudewicz 1
PMCID: PMC1384680  PMID: 1937565

Abstract

We have recently shown that monolayer cultures of calf pulmonary artery endothelial (CPAE) cells pretreated with phorbol myristate acetate (PMA) generate a conditioned medium that is chemotactic for human polymorphonuclear leucocytes (PMNL). Fibronectin (Fn) is a multidomain protein found in the plasma and subendothelial extracellular matrix that induces attachment and migration of a variety of cell types. The present study was designed to evaluate the role of Fn or fragments of Fn present in conditioned medium from phorbol ester-stimulated endothelial cells as potential chemotactic factors for human PMNL. A large number of Fn fragments were revealed by Western immunoblotting of serum-free conditioned medium 4 hr after treatment of CPAE monolayers with PMA. Gelatin-Sepharose affinity chromatography of 4-hr conditioned medium demonstrated chemotactic activity for PMNL in both gelatin-binding and non-gelatin-binding fractions. The addition of bovine Fn antiserum to the conditioned medium inhibited PMNL chemotaxis in a dose-dependent manner while having no effect on PMNL chemotaxis generated by zymosan-activated serum. One site on the Fn molecule known to interact with phagocytic cells is the cell-binding domain containing the Arg-Gly-Asp (RGD) sequence. Pretreatment of PMNL with a RGD-containing peptide (1 mM GRGDSPK) for 10 min completely inhibited the expression of chemotactic activity present in conditioned medium and in the gelatin-binding and non-gelatin-binding fractions. PMNL chemotaxis was not stimulated by either intact Fn or the RGD-containing septapeptide tested over a wide concentration range. However, incubation of PMNL with a purified 120,000-MW fragment of Fn containing the cell-binding domain stimulated chemotaxis in a dose-dependent manner. In contrast, a purified 45,000 MW fragment of Fn containing the gelatin-binding domain was not chemotactic for PMNL. When a monoclonal antibody directed against the cell-binding domain of Fn was incubated with conditioned medium, a significant reduction in PMNL chemotaxis was observed. These results demonstrate that phorbol ester-stimulated pulmonary artery endothelial cells release Fn fragments and suggest an important role for Fn fragments containing the cell-binding domain in stimulating the migration of PMNL.

Full text

PDF
114

Images in this article

117Figure 3
on p.117

Selected References

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

  1. Albini A., Allavena G., Melchiori A., Giancotti F., Richter H., Comoglio P. M., Parodi S., Martin G. R., Tarone G. Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor. J Cell Biol. 1987 Oct;105(4):1867–1872. doi: 10.1083/jcb.105.4.1867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arnaout M. A., Todd R. F., 3rd, Dana N., Melamed J., Schlossman S. F., Colten H. R. Inhibition of phagocytosis of complement C3- or immunoglobulin G-coated particles and of C3bi binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (Mol). J Clin Invest. 1983 Jul;72(1):171–179. doi: 10.1172/JCI110955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Birdwell C. R., Gospodarowicz D., Nicolson G. L. Identification, localization, and role of fibronectin in cultured bovine endothelial cells. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3273–3277. doi: 10.1073/pnas.75.7.3273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bohnsack J. F., Kleinman H. K., Takahashi T., O'Shea J. J., Brown E. J. Connective tissue proteins and phagocytic cell function. Laminin enhances complement and Fc-mediated phagocytosis by cultured human macrophages. J Exp Med. 1985 May 1;161(5):912–923. doi: 10.1084/jem.161.5.912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bowersox J. C., Sorgente N. Chemotaxis of aortic endothelial cells in response to fibronectin. Cancer Res. 1982 Jul;42(7):2547–2551. [PubMed] [Google Scholar]
  6. Brown E. J., Goodwin J. L. Fibronectin receptors of phagocytes. Characterization of the Arg-Gly-Asp binding proteins of human monocytes and polymorphonuclear leukocytes. J Exp Med. 1988 Mar 1;167(3):777–793. doi: 10.1084/jem.167.3.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bryant G., Rao C. N., Brentani M., Martins W., Lopes J. D., Martin S. E., Liotta L. A., Schiffmann E. A role for the laminin receptor in leukocyte chemotaxis. J Leukoc Biol. 1987 Mar;41(3):220–227. doi: 10.1002/jlb.41.3.220. [DOI] [PubMed] [Google Scholar]
  8. Burnett D., Chamba A., Hill S. L., Stockley R. A. Neutrophils from subjects with chronic obstructive lung disease show enhanced chemotaxis and extracellular proteolysis. Lancet. 1987 Nov 7;2(8567):1043–1046. doi: 10.1016/s0140-6736(87)91476-0. [DOI] [PubMed] [Google Scholar]
  9. Clark R. A., Quinn J. H., Winn H. J., Lanigan J. M., Dellepella P., Colvin R. B. Fibronectin is produced by blood vessels in response to injury. J Exp Med. 1982 Aug 1;156(2):646–651. doi: 10.1084/jem.156.2.646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Clark R. A., Wikner N. E., Doherty D. E., Norris D. A. Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment. J Biol Chem. 1988 Aug 25;263(24):12115–12123. [PubMed] [Google Scholar]
  11. Elgebaly S. A., Herkert N., O'Rourke J., Kreutzer D. L. Characterization of neutrophil and monocyte specific chemotactic factors derived from the cornea in response to hydrogen peroxide injury. Am J Pathol. 1987 Jan;126(1):40–50. [PMC free article] [PubMed] [Google Scholar]
  12. Garcia-Pardo A., Ferreira O. C., Valinsky J., Bianco C. Fibronectin receptors of mononuclear phagocytes: binding characteristics and biochemical isolation. Exp Cell Res. 1989 Apr;181(2):420–431. doi: 10.1016/0014-4827(89)90099-2. [DOI] [PubMed] [Google Scholar]
  13. Granelli-Piperno A., Vassalli J. D., Reich E. Secretion of plasminogen activator by human polymorphonuclear leukocytes. Modulation by glucocorticoids and other effectors. J Exp Med. 1977 Dec 1;146(6):1693–1706. doi: 10.1084/jem.146.6.1693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gresham H. D., Goodwin J. L., Allen P. M., Anderson D. C., Brown E. J. A novel member of the integrin receptor family mediates Arg-Gly-Asp-stimulated neutrophil phagocytosis. J Cell Biol. 1989 May;108(5):1935–1943. doi: 10.1083/jcb.108.5.1935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gross J. L., Moscatelli D., Jaffe E. A., Rifkin D. B. Plasminogen activator and collagenase production by cultured capillary endothelial cells. J Cell Biol. 1982 Dec;95(3):974–981. doi: 10.1083/jcb.95.3.974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gudewicz P. W., Odekon L. E., Del Vecchio P. J., Saba T. M. Generation of neutrophil chemotactic activity by phorbol ester-stimulated calf pulmonary artery endothelial cells. J Leukoc Biol. 1988 Jul;44(1):1–7. doi: 10.1002/jlb.44.1.1. [DOI] [PubMed] [Google Scholar]
  17. Gudewicz P. W., Weaver M. B., Del Vecchio P. J., Saba T. M. Phorbol myristate acetate-treated endothelium stimulates polymorphonuclear leukocyte adhesion and superoxide secretion. J Lab Clin Med. 1989 Jun;113(6):708–716. [PubMed] [Google Scholar]
  18. Hosein B., Bianco C. Monocyte receptors for fibronectin characterized by a monoclonal antibody that interferes with receptor activity. J Exp Med. 1985 Jul 1;162(1):157–170. doi: 10.1084/jem.162.1.157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hynes R. O. Integrins: a family of cell surface receptors. Cell. 1987 Feb 27;48(4):549–554. doi: 10.1016/0092-8674(87)90233-9. [DOI] [PubMed] [Google Scholar]
  20. Jaffe E. A., Mosher D. F. Synthesis of fibronectin by cultured human endothelial cells. J Exp Med. 1978 Jun 1;147(6):1779–1791. doi: 10.1084/jem.147.6.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Laskin D. L., Kimura T., Sakakibara S., Riley D. J., Berg R. A. Chemotactic activity of collagen-like polypeptides for human peripheral blood neutrophils. J Leukoc Biol. 1986 Mar;39(3):255–266. doi: 10.1002/jlb.39.3.255. [DOI] [PubMed] [Google Scholar]
  22. Macarak E. J., Kirby E., Kirk T., Kefalides N. A. Synthesis of cold-insoluble globulin by cultured calf endothelial cells. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2621–2625. doi: 10.1073/pnas.75.6.2621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McCarthy J. B., Hagen S. T., Furcht L. T. Human fibronectin contains distinct adhesion- and motility-promoting domains for metastatic melanoma cells. J Cell Biol. 1986 Jan;102(1):179–188. doi: 10.1083/jcb.102.1.179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McNeil P. L., Muthukrishnan L., Warder E., D'Amore P. A. Growth factors are released by mechanically wounded endothelial cells. J Cell Biol. 1989 Aug;109(2):811–822. doi: 10.1083/jcb.109.2.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mercandetti A. J., Lane T. A., Colmerauer M. E. Cultured human endothelial cells elaborate neutrophil chemoattractants. J Lab Clin Med. 1984 Sep;104(3):370–380. [PubMed] [Google Scholar]
  26. Mignatti P., Mazzieri R., Rifkin D. B. Expression of the urokinase receptor in vascular endothelial cells is stimulated by basic fibroblast growth factor. J Cell Biol. 1991 Jun;113(5):1193–1201. doi: 10.1083/jcb.113.5.1193. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Norris D. A., Clark R. A., Swigart L. M., Huff J. C., Weston W. L., Howell S. E. Fibronectin fragment(s) are chemotactic for human peripheral blood monocytes. J Immunol. 1982 Oct;129(4):1612–1618. [PubMed] [Google Scholar]
  28. O'Brien R. F., Seton M. P., Makarski J. S., Center D. M., Rounds S. Thiourea causes endothelial cells in tissue culture to produce neutrophil chemoattractant activity. Am Rev Respir Dis. 1984 Jul;130(1):103–109. doi: 10.1164/arrd.1984.130.1.103. [DOI] [PubMed] [Google Scholar]
  29. Pommier C. G., O'Shea J., Chused T., Yancey K., Frank M. M., Takahashi T., Brown E. J. Studies on the fibronectin receptors of human peripheral blood leukocytes. Morphologic and functional characterization. J Exp Med. 1984 Jan 1;159(1):137–151. doi: 10.1084/jem.159.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ruoslahti E., Pierschbacher M. D. Arg-Gly-Asp: a versatile cell recognition signal. Cell. 1986 Feb 28;44(4):517–518. doi: 10.1016/0092-8674(86)90259-x. [DOI] [PubMed] [Google Scholar]
  31. Sato Y., Rifkin D. B. Autocrine activities of basic fibroblast growth factor: regulation of endothelial cell movement, plasminogen activator synthesis, and DNA synthesis. J Cell Biol. 1988 Sep;107(3):1199–1205. doi: 10.1083/jcb.107.3.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Seppä H. E., Yamada K. M., Seppä S. T., Silver M. H., Kleinman H. K., Schiffmann E. The cell binding fragment of fibronectin is chemotactic for fibroblasts. Cell Biol Int Rep. 1981 Aug;5(8):813–819. doi: 10.1016/0309-1651(81)90253-8. [DOI] [PubMed] [Google Scholar]
  33. Shoji S., Ertl R. F., Linder J., Romberger D. J., Rennard S. I. Bronchial epithelial cells produce chemotactic activity for bronchial epithelial cells. Possible role for fibronectin in airway repair. Am Rev Respir Dis. 1990 Jan;141(1):218–225. doi: 10.1164/ajrccm/141.1.218. [DOI] [PubMed] [Google Scholar]
  34. Sibille Y., Reynolds H. Y. Macrophages and polymorphonuclear neutrophils in lung defense and injury. Am Rev Respir Dis. 1990 Feb;141(2):471–501. doi: 10.1164/ajrccm/141.2.471. [DOI] [PubMed] [Google Scholar]
  35. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wachtfogel Y. T., Abrams W., Kucich U., Weinbaum G., Schapira M., Colman R. W. Fibronectin degradation products containing the cytoadhesive tetrapeptide stimulate human neutrophil degranulation. J Clin Invest. 1988 May;81(5):1310–1316. doi: 10.1172/JCI113456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wright S. D., Craigmyle L. S., Silverstein S. C. Fibronectin and serum amyloid P component stimulate C3b- and C3bi-mediated phagocytosis in cultured human monocytes. J Exp Med. 1983 Oct 1;158(4):1338–1343. doi: 10.1084/jem.158.4.1338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yamada K. M., Olden K. Fibronectins--adhesive glycoproteins of cell surface and blood. Nature. 1978 Sep 21;275(5677):179–184. doi: 10.1038/275179a0. [DOI] [PubMed] [Google Scholar]
  39. Zimmerman G. A., McIntyre T. M. Neutrophil adherence to human endothelium in vitro occurs by CDw18 (Mo1, MAC-1/LFA-1/GP 150,95) glycoprotein-dependent and -independent mechanisms. J Clin Invest. 1988 Feb;81(2):531–537. doi: 10.1172/JCI113351. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Immunology are provided here courtesy of British Society for Immunology

RESOURCES