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. 1994 Nov 2;127(4):1139–1147. doi: 10.1083/jcb.127.4.1139

Complement receptor 3 (CR3, Mac-1, integrin alpha M beta 2, CD11b/CD18) is required for tyrosine phosphorylation of paxillin in adherent and nonadherent neutrophils

PMCID: PMC2200044  PMID: 7525604

Abstract

Expression of the leukocyte (beta 2) integrins is required for many functions of activated neutrophils (PMN), even when there is no recognized ligand for any beta 2 integrin. To investigate the hypothesis that beta 2 integrins may be involved in a signal transduction pathway related to cytoskeletal reorganization, we examined whether beta 2 integrins have a role in tyrosine phosphorylation of the cytoskeletal protein paxillin. Treatment of PMN in suspension with phorbol esters, f-Met-Leu-Phe, and TNF-alpha resulted in paxillin tyrosine phosphorylation. However, treatment of beta 2-deficient (LAD) PMN failed to induce paxillin tyrosine phosphorylation. Normal PMN phosphorylated paxillin in response to adhesion to immune complexes, while the LAD PMN did not. Adhesion of phorbol ester activated-LAD PMN to the extracellular matrix proteins fibronectin, laminin, and vitronectin failed to induce paxillin tyrosine phosphorylation. Treatment of activated normal PMN with mAb directed against the beta 2 integrin alpha chains demonstrated that CR3 (alpha M beta 2) was required for paxillin phosphorylation. Transfection of the cell line K562 with CR3 confirmed that CR3 ligation resulted in paxillin tyrosine phosphorylation. As a control, K562 transfected with CR2 (CD21) which bound equally avidly to the same complement C3-derived ligand (C3bi) as the CR3 transfectants, showed no enhanced tyrosine phosphorylation of paxillin upon receptor ligation. While both CR2 and CR3 transfectants showed efficient adhesion to a C3bi-coated surface, only the CR3 transfectants spread during adhesion and phosphorylated paxillin. Together these data demonstrate that CR3 is required for paxillin phosphorylation during activation of both adherent and nonadherent PMN. Even PMN activated in suspension or by adhesion to immune complexes, when no CR3 ligand is apparent, still require CR3 for a signal transduction pathway leading to paxillin tyrosine phosphorylation. This pathway is likely to be important for PMN function in inflammation and host defense.

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Selected References

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  1. Anderson D. C., Schmalstieg F. C., Arnaout M. A., Kohl S., Tosi M. F., Dana N., Buffone G. J., Hughes B. J., Brinkley B. R., Dickey W. D. Abnormalities of polymorphonuclear leukocyte function associated with a heritable deficiency of high molecular weight surface glycoproteins (GP138): common relationship to diminished cell adherence. J Clin Invest. 1984 Aug;74(2):536–551. doi: 10.1172/JCI111451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berkow R. L., Wang D., Larrick J. W., Dodson R. W., Howard T. H. Enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor. J Immunol. 1987 Dec 1;139(11):3783–3791. [PubMed] [Google Scholar]
  3. Bockholt S. M., Burridge K. Cell spreading on extracellular matrix proteins induces tyrosine phosphorylation of tensin. J Biol Chem. 1993 Jul 15;268(20):14565–14567. [PubMed] [Google Scholar]
  4. Bohnsack J. F., Akiyama S. K., Damsky C. H., Knape W. A., Zimmerman G. A. Human neutrophil adherence to laminin in vitro. Evidence for a distinct neutrophil integrin receptor for laminin. J Exp Med. 1990 Apr 1;171(4):1221–1237. doi: 10.1084/jem.171.4.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bohnsack J. F. CD11/CD18-independent neutrophil adherence to laminin is mediated by the integrin VLA-6. Blood. 1992 Mar 15;79(6):1545–1552. [PubMed] [Google Scholar]
  6. Brown E. J., Bohnsack J. F., Gresham H. D. Mechanism of inhibition of immunoglobulin G-mediated phagocytosis by monoclonal antibodies that recognize the Mac-1 antigen. J Clin Invest. 1988 Feb;81(2):365–375. doi: 10.1172/JCI113328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burridge K., Turner C. E., Romer L. H. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol. 1992 Nov;119(4):893–903. doi: 10.1083/jcb.119.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carel J. C., Frazier B., Ley T. J., Holers V. M. Analysis of epitope expression and the functional repertoire of recombinant complement receptor 2 (CR2/CD21) in mouse and human cells. J Immunol. 1989 Aug 1;143(3):923–930. [PubMed] [Google Scholar]
  9. Davignon D., Martz E., Reynolds T., Kürzinger K., Springer T. A. Lymphocyte function-associated antigen 1 (LFA-1): a surface antigen distinct from Lyt-2,3 that participates in T lymphocyte-mediated killing. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4535–4539. doi: 10.1073/pnas.78.7.4535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Diamond M. S., Staunton D. E., de Fougerolles A. R., Stacker S. A., Garcia-Aguilar J., Hibbs M. L., Springer T. A. ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18). J Cell Biol. 1990 Dec;111(6 Pt 2):3129–3139. doi: 10.1083/jcb.111.6.3129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dransfield I., Cabañas C., Craig A., Hogg N. Divalent cation regulation of the function of the leukocyte integrin LFA-1. J Cell Biol. 1992 Jan;116(1):219–226. doi: 10.1083/jcb.116.1.219. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Fuhlbrigge R. C., Fine S. M., Unanue E. R., Chaplin D. D. Expression of membrane interleukin 1 by fibroblasts transfected with murine pro-interleukin 1 alpha cDNA. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5649–5653. doi: 10.1073/pnas.85.15.5649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fuortes M., Jin W. W., Nathan C. Adhesion-dependent protein tyrosine phosphorylation in neutrophils treated with tumor necrosis factor. J Cell Biol. 1993 Feb;120(3):777–784. doi: 10.1083/jcb.120.3.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Glenney J. R., Jr, Zokas L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol. 1989 Jun;108(6):2401–2408. doi: 10.1083/jcb.108.6.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Graham I. L., Brown E. J. Extracellular calcium results in a conformational change in Mac-1 (CD11b/CD18) on neutrophils. Differentiation of adhesion and phagocytosis functions of Mac-1. J Immunol. 1991 Jan 15;146(2):685–691. [PubMed] [Google Scholar]
  16. Graham I. L., Lefkowith J. B., Anderson D. C., Brown E. J. Immune complex-stimulated neutrophil LTB4 production is dependent on beta 2 integrins. J Cell Biol. 1993 Mar;120(6):1509–1517. doi: 10.1083/jcb.120.6.1509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Greenberg S., Chang P., Silverstein S. C. Tyrosine phosphorylation of the gamma subunit of Fc gamma receptors, p72syk, and paxillin during Fc receptor-mediated phagocytosis in macrophages. J Biol Chem. 1994 Feb 4;269(5):3897–3902. [PubMed] [Google Scholar]
  18. Gresham H. D., Clement L. T., Lehmeyer J. E., Griffin F. M., Jr, Volanakis J. E. Stimulation of human neutrophil Fc receptor-mediated phagocytosis by a low molecular weight cytokine. J Immunol. 1986 Aug 1;137(3):868–875. [PubMed] [Google Scholar]
  19. Gresham H. D., Graham I. L., Anderson D. C., Brown E. J. Leukocyte adhesion-deficient neutrophils fail to amplify phagocytic function in response to stimulation. Evidence for CD11b/CD18-dependent and -independent mechanisms of phagocytosis. J Clin Invest. 1991 Aug;88(2):588–597. doi: 10.1172/JCI115343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Heffetz D., Bushkin I., Dror R., Zick Y. The insulinomimetic agents H2O2 and vanadate stimulate protein tyrosine phosphorylation in intact cells. J Biol Chem. 1990 Feb 15;265(5):2896–2902. [PubMed] [Google Scholar]
  21. Huang M. M., Lipfert L., Cunningham M., Brugge J. S., Ginsberg M. H., Shattil S. J. Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK. J Cell Biol. 1993 Jul;122(2):473–483. doi: 10.1083/jcb.122.2.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Järvinen M., Ylänne J., Virtanen I. The effect of differentiation inducers on the integrin expression of K562 erythroleukemia cells. Cell Biol Int. 1993 Apr;17(4):399–407. doi: 10.1006/cbir.1993.1078. [DOI] [PubMed] [Google Scholar]
  23. Malhotra V., Hogg N., Sim R. B. Ligand binding by the p150,95 antigen of U937 monocytic cells: properties in common with complement receptor type 3 (CR3). Eur J Immunol. 1986 Sep;16(9):1117–1123. doi: 10.1002/eji.1830160915. [DOI] [PubMed] [Google Scholar]
  24. Todd R. F., 3rd, Nadler L. M., Schlossman S. F. Antigens on human monocytes identified by monoclonal antibodies. J Immunol. 1981 Apr;126(4):1435–1442. [PubMed] [Google Scholar]
  25. Turner C. E., Glenney J. R., Jr, Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol. 1990 Sep;111(3):1059–1068. doi: 10.1083/jcb.111.3.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Turner C. E. Paxillin: a cytoskeletal target for tyrosine kinases. Bioessays. 1994 Jan;16(1):47–52. doi: 10.1002/bies.950160107. [DOI] [PubMed] [Google Scholar]
  27. Van Strijp J. A., Russell D. G., Tuomanen E., Brown E. J., Wright S. D. Ligand specificity of purified complement receptor type three (CD11b/CD18, alpha m beta 2, Mac-1). Indirect effects of an Arg-Gly-Asp (RGD) sequence. J Immunol. 1993 Sep 15;151(6):3324–3336. [PubMed] [Google Scholar]
  28. Volpp B. D., Nauseef W. M., Donelson J. E., Moser D. R., Clark R. A. Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase. Proc Natl Acad Sci U S A. 1989 Sep;86(18):7195–7199. doi: 10.1073/pnas.86.18.7195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Weng Z., Taylor J. A., Turner C. E., Brugge J. S., Seidel-Dugan C. Detection of Src homology 3-binding proteins, including paxillin, in normal and v-Src-transformed Balb/c 3T3 cells. J Biol Chem. 1993 Jul 15;268(20):14956–14963. [PubMed] [Google Scholar]
  30. Woods A., Couchman J. R. Protein kinase C involvement in focal adhesion formation. J Cell Sci. 1992 Feb;101(Pt 2):277–290. doi: 10.1242/jcs.101.2.277. [DOI] [PubMed] [Google Scholar]
  31. Wright S. D., Meyer B. C. Phorbol esters cause sequential activation and deactivation of complement receptors on polymorphonuclear leukocytes. J Immunol. 1986 Mar 1;136(5):1759–1764. [PubMed] [Google Scholar]
  32. Wright S. D., Rao P. E., Van Voorhis W. C., Craigmyle L. S., Iida K., Talle M. A., Westberg E. F., Goldstein G., Silverstein S. C. Identification of the C3bi receptor of human monocytes and macrophages by using monoclonal antibodies. Proc Natl Acad Sci U S A. 1983 Sep;80(18):5699–5703. doi: 10.1073/pnas.80.18.5699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Zachary I., Sinnett-Smith J., Turner C. E., Rozengurt E. Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of the focal adhesion-associated protein paxillin in Swiss 3T3 cells. J Biol Chem. 1993 Oct 15;268(29):22060–22065. [PubMed] [Google Scholar]

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