Measurement of VLA‐4/CS‐1 and VLA‐4/VCAM Adhesion Inhibition

Christopher Mehlin1

1 University of Washington, Seattle, Washington
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.7
DOI:  10.1002/0471141755.ph1207s24
Online Posting Date:  May, 2004
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Abstract

Cell adhesion, a critical early step in the inflammatory process, has increasingly become the target of drug discovery efforts. Described in this unit are techniques for measuring inhibitors of VLA-4-mediated adhesion to either VCAM or the connecting segment (CS-1) of fibronectin.

Keywords: VLA-4; VCAM; CS-1; cell adhesion; inflammation

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol: Measurement of VLA-4/CS-1 Adhesion Inhibition
  • Alternate Protocol: Measurement of VLA-4/VCAM Adhesion Inhibition
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol: Measurement of VLA-4/CS-1 Adhesion Inhibition

 Materials
  • Biotinylated CS-1 peptide: biotin-(CH2)3CO-GPEILDVPST-NH3
  • DPBS with calcium and magnesium (Life Technologies)
  • BSA (e.g., Sigma)
  • Binding buffer (see recipe)
  • Test compounds
  • Cyclic control peptide (C-ss-CWLDV)
  • RAMOS cells (ATCC #CRL-1596), grown in RPMI with 10% FBS and 10 mM HEPES
  • 1 µg/µl calcein-AM (Molecular Probes) in DMSO
  • Lysis buffer (see recipe)
  • 96-well Neutravidin-coated plates (Pierce)
  • Gentle ELISA plate–washing apparatus (e.g., Molecular Devices' Skanwasher 400; for automatic washing) or 12-channel pipettor and tips (for manual washing)
  • 96-well polypropylene plates
  • Fluorescence plate reader (e.g., BioTek Synergy HT)
  • Nonlinear regression software (e.g., Prism, GraphPad)
  • Additional reagents and equipment for counting cells and measuring viability (Phelan, 1997)

Alternate Protocol: Measurement of VLA-4/VCAM Adhesion Inhibition

 Additional Materials (also see Basic Protocol)
  • HUVEC cells (e.g., Clonetics)
  • HUVEC medium (e.g., EGM from Clonetics)
  • TNF- (e.g., Calbiochem)
  • 96-well tissue culture plates
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Figures

  •  FigureFigure 12.7.1 Standard RAMOS binding assay to CS-1 peptide and inhibition by cyclic peptide. The histogram on the left illustrates the binding of RAMOS cells to the Neutravidin-CS-1 complex in the absence of an inhibitor or cyclic peptide (hatched bar) or to Neutravidin alone (solid bar). All values are five replicates ±SEM.
    View Image
  •  FigureFigure 12.7.2 Standard expected results using RAMOS cells binding to TNF--stimulated HUVEC cells and inhibition by cyclic peptide. The histogram on the left illustrates the binding of RAMOS cells to TNF-stimulated HUVEC cells (solid bar) and to HUVEC cells which were not stimulated with TNF (hatched bar). All values are five replicates ±SEM.
    View Image

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Literature Cited

Literature Cited
    Hocking, D.C. 2002. Fibronectin matrix deposition and cell contractility. Chest 122:275S-278S.
    Lin, K., Ateeq, H.S., Hsiung, S.H., Chong, L.T., Zimmerman, C.N., Castro, A., Lee, W.C., Hammond, C.E., Kalkunte, S., Chen, L.L., Pepinsky, R.B., Leone, D.R., Sprague, A.G., Abraham, W.M., Gill, A., Lobb, R.R., Adams, S.P. 1999. Selective, tight-binding inhibitors of integrin 41 that inhibit allergic airway responses. J. Med. Chem. 42(5):920-34.
    Lobb, R.R. and Adams, S.P. 1999. Small molecule antagonists of 4 integrins: Novel drugs for asthma. Exp. Opin. Invest. Drugs. 8(7):935-945.
    Lobb, R.R., Abraham, W.M., Burkly, L.C., Gill, A., Ma, W., Knight, J.A., Leone, D.R., Antognetti, G., and Pepinsky, R.B. 1996. Pathophysiologic role of alpha4 integrins in the lung. Ann. N.Y. Acad. Sci. 796:113-123.
    Masumoto, A. and Hemler, M.E. 1993. Multiple activation states of VLA-4. J. Biol. Chem. 268:228-234.
    Phelan, M.C. 1997. Techniques for mammalian cell tissue culture. In Current Protocols in Protein Science (J.E. Coligan, B.M. Dunn, D.W. Speicher, P.T. Wingfield, eds.) pp. A.3C.1-A.3C.14. John Wiley & Sons, Hoboken, N.J.
    Swerlick, R.A., Lee, K.H., Li, L., Sepp, N.T., Caughman, S.W. and Lawley, T.J. 1992. Regulation of vascular cell adhesion molecule 1 on human dermal microvascular endothelial cells. J. Immunol. 149:698-705.
    van der Flier, A. and Sonnenberg, A. 2001. Function and interactions of integrins. Cell Tissue Res. 305:285-298.
    Vanderslice, P. Ren, K., Revelle, J.K., Kim, D.C., Scott, D., Bjercke, R.J., Yeh, E.T.H., Beck, P.J., and Kogan, T.P. 1997. A cyclic hexapeptide is a potent antagonist of 4 integrins. J. Immunol. 158:1710-1718.
    Yang, G.X. and Hagman, W.K. 2003. VLA-4 antagonists: Potent inhibitors of lymphocyte migration. Med. Res. Rev. 23:369-392.
    Yusuf-Makagiansar, H., Anderson, M.E., Yakovleva, T.V., Murray, J.S., and Siahaan, T.J. 2002. Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic approach to inflammation and autoimmune disease. Med. Res. Rev. 22(2):146-67.
 Key Reference
    Yusuf-Makagiansar et al., 2002. See above.

This is a broad review covering cell adhesion as a therapeutic target with specific reference to VLA-4.

    Yang et al., 2003. See above.

This review focuses on compounds developed as VLA-4 inhibitors.

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