Mechanism of binding and internalization of ICAM-1-derived cyclic peptides by LFA-1 on the surface of T cells: a potential method for targeted drug delivery.
OBJECTIVE
Peptides derived from the Domain 1 of the adhesion molecule ICAM-1(1-21) are being developed as targeting ligands for LFA-1 receptors expressed on activated T cells. This work aims to elucidate the binding and internalization of ICAM-1-derived cyclic peptides (cIBL, cIBC, and cIBR) to LFA-1.


METHODS
Ninety-six-well plates coated with soluble LFA-1 (sLFA-1) were used to characterize the binding of FITC-labeled peptide. An anti-CD11a antibody to the I-domain of LFA-1 was used to inhibit the binding of these peptides, which was quantified using a fluorescence plate reader. An unrelated FITC-labeled cyclic peptide was used as a negative control, and PE-labeled anti-CD11a antibodies (PE-R3.2 and PE-R7.1) were used as positive controls. Peptide binding to cell surface LFA-1 was visualized using colocalization of FITC-cIBR peptide and PE-labeled anti-CD18 antibody (LFA-1 beta-subunit) on SKW-3 T cells by fluorescent microscopy. Inhibition of ICAM-1 binding to LFA-1 by peptides was evaluated using a Biacore assay. Binding and internalization of FITC-labeled peptides were evaluated by flow cytometry and confocal microscopy at 4 degrees C and 37 degrees C.


RESULTS
These FITC-labeled cyclic peptides bind to sLFA-1 and can be blocked by an anti-CD11a antibody to the I-domain, suggesting that their binding site is on the I-domain of LFA-1. The FITC-cIBR peptide was localized with an anti-CD18 antibody on the surface of T cells, indicating that the FITC-cIBR peptide binds to LFA-1 on the cell surface. Flow cytometry and confocal microscopy demonstrated that FITC-labeled peptides were internalized in a temperature-dependent manner. Biacore analysis demonstrated that these peptides did not inhibit sICAM-1 from binding to immobilized sLFA-1. However, the binding properties of the soluble forms of LFA-1 and ICAM-1 may not correlate to their interaction at the cell surface.


CONCLUSIONS
Cyclic ICAM-1-derived peptides (cIBL, cIBC, and cIBR) bind to the I-domain of LFA-1 and are internalized by LFA-1 receptors on the surface of T cells. Therefore, these peptides could be used to target and deliver drugs to the cytoplasmic domain of T cells.
Anderson
Meagan E
ME
Department of Pharmaceutical Chemistry, The University of Kansas, Simons Research Laboratories, Lawrence, Kansas 66047, USA.
Siahaan
Teruna J
TJ
D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D016650:Fluorescein-5-isothiocyanate; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000737:chemistry / Q000378:metabolism*; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D018613:Microscopy, Confocal; D010456:Peptides, Cyclic / Q000737:chemistry / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D013601:T-Lymphocytes / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors
D016169:Lymphocyte Function-Associated Antigen-1; D010456:Peptides, Cyclic; D011948:Receptors, Antigen, T-Cell; D018799:Intercellular Adhesion Molecule-1; D016650:Fluorescein-5-isothiocyanate