Binding and Internalization of an LFA-1-Derived Cyclic Peptide by ICAM Receptors on Activated Lymphocyte: A Potential Ligand for Drug Targeting to ICAM-1-Expressing Cells
Purpose. The interaction of cell-adhesion molecules LFA-1/ICAM-1 is critical for many inflammatory and immune responses. Blockades of this interaction using antibodies or peptide analogs are being developed as therapeutic approaches for inflammatory and autoimmune diseases. The aim of this study is to examine the binding and internalization mechanisms of LFA-1 peptide [cLAB.L or cyclo-(1,12)-PenITDGEATDSGC] mediated by ICAM receptors on the surface of lymphocytes.
Methods. The binding and internalization of cLAB.L were evaluated using fluorescence-labeled cLAB.L on activated Molt-3 cells, measured by flow cytometry. Confocal fluorescence microscopy was also used to image the distribution of peptide binding and internalization.
Results. The binding of FITC-cLAB.L exhibited bimodal cell distribution and was enhanced by Ca2+ and Mg2+. Marked differences in peptide binding were found between 37 and 4°C, as well as between activated and non-activated cells. Unlabeled peptide, low temperature, and the absence of cell activation suppress the peptide binding. The presence of peptide in the cytoplasm was detected in 37 but not 4°C binding. Peptide cLAB.L inhibited the binding of monoclonal antibodies to domain D1 of ICAM-1 and domain D1 of ICAM-3.
Conclusions. Peptide cLAB.L can bind to the D1-domain of ICAM-1 and, to a lesser extent, to ICAM-3 on activated T-cells. Peptide binding indicates responses to the multiple and dynamic states of activated receptor ICAMs; this peptide may also be internalized by ICAM receptors on T-cells. This work suggests that cLAB.L has a therapeutic potential to target drugs to ICAM-1 expressing cells including autoreactive lymphocytes and inflamed tissues.
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- 1.C. Wülfing and M. M. Davis. A receptor/cytoskeletal movement triggered by costimulation during T cell activation. Science 282:2266-2269 (1998).Google Scholar
- 2.A. R. de Fougerolles, X. Qin, and T. A. Springer. Characterization of the function of intercellular adhesion molecule (ICAM)-3 and comparison with ICAM-1 and ICAM-2 in immune responses. J. Exp. Med. 179:619-629 (1994).Google Scholar
- 3.R. Rothlein and T. A. Springer. The requirement for lymphocyte function associated antigen-1 in homotypic leukocyte adhesion stimulated by phorbol ester. J. Exp. Med. 163:1132-1149 (1986).Google Scholar
- 4.Y. van Kooyk, P. Weder, K. Heije, R. De Waal Malefijt, and C. G. Figdor. Critical amino acids in the lymphocyte function-associated antigen-1 I domain mediate intercellular adhesion molecule 3 binding and immune function. Cell Adhesion. Comm. 1:21-32 (1993).Google Scholar
- 5.P. Stanley and N. Hogg. The I-domain of integrin LFA-1 interacts with ICAM-1 domain-1 at residue glu-34 but not gln-73. J. Biol. Chem. 273:3358-3362 (1998).Google Scholar
- 6.E. K. Nakamura, R. A. Shorthouse, B. Zheng, S. M. McCabe, P. M. Jardieu, and R. E. Morris. Long-term survival of solid organ allografts by brief anti-lymphocyte function-associated antigen-1 monoclonal monotherapy. Transplantation 62:547-552 (1996).Google Scholar
- 7.L. S. Davis, A. F. Kavanaugh, L. A. Nichols, and P. E. Lipsky. Induction of persistent T cell hyporesponsiveness in vivo by monoclonal antibody to ICAM-1 in patients with rheumatoid arthritis. J. Immunol. 154:3525-3537 (1995).Google Scholar
- 8.D. O. Willenborg, M. A. Staykova, and M. Miyasaka. Short testament with soluble neuroantigen and anti-CD11a (LFA-1) protects rats against autoimmune encephalomyelitis: Treatment abrogates autoimmune disease but not autoimmunity. J. Immunol. 157:1973-1980 (1996).Google Scholar
- 9.L. Ross, F. Hassman, and L. Molony. Inhibition of Molt-4 endothelial adherence by synthetic peptides from the sequence of ICAM-1. J. Biol. Chem. 267:8537-8543 (1992).Google Scholar
- 10.J. V. Fecondo, N. C. Pavuk, K. A. Silburn, D. M. Y. Read, A. S. Mansell, A. W. Boyd, and D. A. McPhee. Inhibition of Molt-4 endothelial adherence by synthetic peptides from the sequence of ICAM-1. AIDS Res. Hum. Retrovirus. 9:733-740 (1993).Google Scholar
- 11.R. N. Gürsoy and T. J. Siahaan. Binding and internalization of an ICAM-1 peptide by the surface receptors of T-cells. J. Peptide Res. 53:414-421 (1999).Google Scholar
- 12.R. N. Gürsoy, S. D. S. Jois, and T. J. Siahaan. Structural recognition of an ICAM-1 peptide by its receptor on the surface of T cells: conformational studies of cyclo (1,12)-Pen-Pro-Arg-Gly-Gly-Ser-Val-Leu-Val-Thr-Gly-Cys-OH. J. Peptide Res. 53:422-431 (1999).Google Scholar
- 13.T. J. Siahaan, S. A. Tibbets, S. D. S. Jois, M. A. Chan, and S. A. Benedict. Counter receptor-binding domains that block or enhance binding to LFA-1 or ICAM-1. In P. T. P. Kaumaya and R. S. Hodges (eds.), Peptides: Chemistry, Structure and Biology, Mayflower Scientific, England, 1996 pp. 792-793.Google Scholar
- 14.S. H. Benedict, T. J. Siahaan, M. A. Chan, and S. A. Tibbetts. ICAM-1/LFA-1 short-chain peptides and method of using same. US Patent 229, 531 (1994).Google Scholar
- 15.S. A. Tibbetts, C. Chirathaworn, M. Nakashima, S. D. S. Jois, T. J. Siahaan, M. A. Chan, and S. H. Benedict. Peptides derived from ICAM-1 and LFA-1 modulate T cell adhesion and immune function in a mixed lymphocyte culture. Transplantation 68:685-692 (1999).Google Scholar
- 16.T. J. Raub and S. L. Kuentzel. Kinetic and morphological evidence for endocytosis of mammalian cell integrin receptors by using anti-fibronectin receptor beta subunit monoclonal anti-body. Exp. Cell. Res. 184:407-426 (1989).Google Scholar
- 17.A. Almenar-Queralt, A. Duperray, L. A. Miles, and D. C. Altieri. Apical topography and modulation of ICAM-1 expression on activated endothelium. Am. J. Pathol. 147:1278-1288 (1995).Google Scholar
- 18.I. Ricard, M. D. Payet, and G. Dupuis. VCAM-1 is internalized by a clathrin-related pathway in human endothelial cells but its α4β1 integrin counter-receptor remains associated with the plasma membrane in human lymphocytes. Eur. J. Immunol. 28:1708-1718 (1998).Google Scholar
- 19.C. D. Buckley, D. Pilling, N. V. Henriquez, G. Parsonage, K. Threlfall, D. Scheel-Toellner, D. L. Simmons, A. N. Akbar, J. M. Lord, and M. Salmon. Role of ligands in the activation of LFA-1. Nature 397:534-539 (1999).Google Scholar
- 20.S. D. S. Jois, U. S. F. Tambunan, S. Chakrabarti, and T. J. Siahaan. Solution structure of a cyclic RGD peptide that inhibits platelet aggregation. J. Biomol. Struct. Dyn. 14:1-11 (1996).Google Scholar
- 21.P. A. Detmer and S. D. Wright. Adhesion-promoting receptors on leukocytes. Curr. Op. Immunol. 1:10-15 (1988).Google Scholar
- 22.E. Zisman, Y. Katz-Levy, M. Dayan, S. L. Kirshner, M. Paas-Rozner, A. Karni, O. Abramsky, C. Brautbar, M. Fridkins, M. Sela, and E. Mozes. Peptide analogs to pathogenic epitopes of the human acetylcholine receptor α subunit as potential modulators of myasthenia gravis. Proc. Natl. Acad. Sci. USA 93:4492-4497 (1996).Google Scholar
- 23.P. L. Reilly, J. R. Woska Jr., D. D. Jeanfavre, E. McNally, R. Rothlein, and B. J. Bormann. The native structure of intercellular adhesion molecule-1 (ICAM-1) is a dimer. Correlation with binding to LFA-1. J. Immunol. 155:529-532 (1995).Google Scholar
- 24.C. Huang and T. A. Springer. A binding interface on the I domain of lymphocyte function-associated antigen-1 (LFA-1) required for specific interaction with intercellular adhesion molecule (ICAM-1). J. Biol. Chem. 270:19008-19016 (1995).Google Scholar
- 25.C. P. Edwards, K. L. Fisher, L. G. Presta, and S. C. Bodary. Mapping the intercellular adhesion molecule-1 and-3 binding site on the inserted domain of leukocyte function-associated antigen-1. J. Biol. Chem. 273:28937-28944 (1998).Google Scholar
- 26.M. E. Binnerts, Y. van Kooyk, C. P. Edwards, M. Champe, L. Presta, S. C. Bodary, C. G. Figdor, and P. W. Berman. Antibodies that selectively inhibit leukocyte function-associated antigen 1 binding to intercellular adhesion molecule-3 recognize a unique epitope within the CD11a I domain. J. Biol. Chem. 271:9962-9968 (1996).Google Scholar
- 27.C. L. Holness, P. A. Bates, A. J. Littler, C. D. Buckley, A. McDowall, D. Bossy, N. Hogg, and D. L. Simmons. Analysis of the binding site on intercellular adhesion molecule 3 for the leukocyte integrin function-associated antigen 1. J. Biol. Chem. 270:877-884 (1995).Google Scholar
- 28.E. D. Bell, A. P. May, and D. L. Simmons. The leukocyte function-associated antigen-1 (LFA-1)-binding site on ICAM-3 comprises residues on both faces of the first immunoglobulin domain. J. Immunol. 161:1363-1370 (1998).Google Scholar
- 29.J. R. Woska, Jr., M. M. Morelock, D. D. Jeanfavre, G. O. Caviness, B. Bormann, and R. Rothlein. Molecular comparison of soluble intercellular adhesion molecule (sICAM)-1 and sICAM-3 binding to lymphocyte function-associated antigen-1. J. Biol. Chem. 273:4725-4733 (1998).Google Scholar
- 30.A. McDowall, B. Leitinger, P. Stanley, P. A. Bates, A. M. Randi, and N. Hogg. The I domain of integrin leukocyte function-associated antigen-1 is involved in a conformational change leading to high affinity binding to ligand intercellular adhesion molecule 1 (ICAM-1). J. Biol. Chem. 273:27396-27403 (1998).Google Scholar
- 31.K. Kurzinger, T. Reynolds, R. N. Germain, D. Davignon, E. Martz, and T. A. Springer. A novel lymphocyte function-associated antigen (LFA-1): Cellular distribution, quantitative expression, and structure. J. Immunol. 127:596-602 (1981).Google Scholar
- 32.A. J. García, J. Takagi, and D. Boettiger. Two-stage activation for α5β1 integrin binding to surface-adsorbed fibronectin. J. Biol. Chem. 273:34710-34715 (1998).Google Scholar