Inhibition of b 2 Integrin-mediated Leukocyte Cell Adhesion by Leucine-Leucine-Glycine Motif-containing Peptides (original) (raw)
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Journal of Cell Biology, 2001
Many integrins mediate cell attachment to the extracellular matrix by recognizing short tripeptide sequences such as arginine-glycine-aspartic acid and leucine-aspartate-valine. Using phage display, we have now found that the leukocyte-specific  2 integrins bind sequences containing a leucine-leucine-glycine (LLG) tripeptide motif. An LLG motif is present on intercellular adhesion molecule (ICAM)-1, the major  2 integrin ligand, but also on several matrix proteins, including von Willebrand factor. We developed a novel  2 integrin antagonist peptide CPCFLLGCC (called LLG-C4), the structure of which was determined by nuclear magnetic resonance. The LLG-C4 peptide inhibited leukocyte ad-hesion to ICAM-1, and, interestingly, also to von Willebrand factor. When immobilized on plastic, the LLG-C4 sequence supported the  2 integrin-mediated leukocyte adhesion, but not  1 or  3 integrin-mediated cell adhesion. These results suggest that LLG sequences exposed on ICAM-1 and on von Willebrand factor at sites of vascular injury play a role in the binding of leukocytes, and LLG-C4 and peptidomimetics derived from it could provide a therapeutic approach to inflammatory reactions.
Inhibition of β2 Integrin–mediated Leukocyte Cell Adhesion
2001
Many integrins mediate cell attachment to the extracellular matrix by recognizing short tripeptide sequences such as arginine-glycine-aspartic acid and leucine-aspartate-valine. Using phage display, we have now found that the leukocyte-specific  2 integrins bind sequences containing a leucine-leucine-glycine (LLG) tripeptide motif. An LLG motif is present on intercellular adhesion molecule (ICAM)-1, the major  2 integrin ligand, but also on several matrix proteins, including von Willebrand factor. We developed a novel  2 integrin antagonist peptide CPCFLLGCC (called LLG-C4), the structure of which was determined by nuclear magnetic resonance. The LLG-C4 peptide inhibited leukocyte adhesion to ICAM-1, and, interestingly, also to von Willebrand factor. When immobilized on plastic, the LLG-C4 sequence supported the  2 integrin-mediated leukocyte adhesion, but not  1 or  3 integrin-mediated cell adhesion. These results suggest that LLG sequences exposed on ICAM-1 and on von Willebrand factor at sites of vascular injury play a role in the binding of leukocytes, and LLG-C4 and peptidomimetics derived from it could provide a therapeutic approach to inflammatory reactions.
Journal of Cell Biology, 1993
Despite the identification and characterization of several distinct ligands for the leukocyte integrin (CD11/CD18) family of adhesion receptors, little is known about the structural regions on these molecules that mediate ligand recognition. In this report, we use alpha subunit chimeras of Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18), and an extended panel of newly generated and previously characterized mAbs specific to the alpha chain of Mac-1 to map the binding sites for four distinct ligands for Mac-1: iC3b, fibrinogen, ICAM-1, and the as-yet uncharacterized counter-receptor responsible for neutrophil homotypic adhesion. Epitopes of mAbs that blocked ligand binding were mapped with the chimeras and used to localize the ligand recognition sites because the data obtained from functional assays with the Mac-1/p150,95 chimeras were not easily interpreted. Results show that the I domain on the alpha chain of Mac-1 is an important recognition site for all four ligands, and that the NH2-...
Journal of Leukocyte Biology
The aggregation of human neutrophils in suspension has features that are analogous to their attachment to activated endothelium in that both involve selectin and  2 -integrin adhesion receptors. For the collisional interaction that forms neutrophil aggregates in suspension, there is a tethering step in which L-selectin on neutrophils binds PSGL-1. At relatively low shear rates (100-200 s -1 ) firm adhesion is mediated in equal measure by LFA-1 binding to ICAM-3, and Mac-1 binding to an as yet undefined ligand. In this report we used a mouse melanoma cell line expressing an estimated 700,000 ICAM-1 (CD54) to examine the relative roles of LFA-1 and Mac-1 over the kinetics of heterotypic cell adhesion in shear mixed suspensions. Neither heterotypic nor homotypic neutrophil aggregates formed with application of shear alone. However, the rate of aggregation peaked within seconds of chemotactic stimulation. In contrast to homotypic aggregation, neither L-selectin nor its O-glycoprotein ligands on neutrophils contributed to heterotypic adhesion. Adhesion was inhibited in a dose-dependent manner as ICAM-1 was titrated with blocking mAb. A direct interaction between LFA-1 and ICAM-1 was preferred over the first minute of stimulation, whereas at later times adhesion was supported equally by Mac-1. Activation with MnCl 2 also favored participation of the constitutively expressed LFA-1. Application of defined shear in a cone and plate viscometer showed that adhesion to the ICAM-1 cells decreased from a maximum level to baseline as shear rate increased up to 400 s -1 in a manner typical of integrin adhesion alone. In contrast, homotypic aggregation supported by the transition from selectin to integrin binding exhibited an increase in efficiency up to 800 s -1 . The pathophysiological significance of receptor site density and duration of contact in collisional interactions relevant to leukocyte recruitment compared to leukocyte-endothelial cell interactions on surfaces is discussed. J. Leukoc. Biol. 64: 622-630; 1998.
Journal of Biological Chemistry, 1998
On T cells the leukocyte integrin leukocyte functionassociated antigen-1 (LFA-1) (CD11a/CD18) can be induced to bind its ligand intercellular adhesion molecule 1 (ICAM-1) (CD54) either by increasing the affinity of the receptor with Mg 2؉ and EGTA or by receptor clustering following activation with phorbol ester. The existence of these two adhesion-inducing pathways implies that alternative mechanisms might exist by which LFA-1 engages ICAM-1. The LFA-1 ␣ subunit I domain contains a major binding site for ICAM-1. In this study we show that soluble LFA-1 I domain blocks ICAM-1 binding of the high affinity Mg 2؉-induced form of LFA-1 but not the phorbol ester-induced form. Under conditions of Mg 2؉activation, the soluble I domain also prevents expression of an activation dependent epitope on LFA-1, implying that it inhibits a conformational change necessary for conversion to the high affinity form of this integrin. In addition, the binding of Mg 2؉-activated LFA-1 to ICAM-1 is blocked by peptides covering the ␣4-3 loop, the 3-␣5 loop, and the ␣5 helix of the I domain, whereas none of the peptides tested blocks phorbol ester-mediated adhesion. The blocking peptides localize to the same face of the crystal structure of the LFA-1 I domain and define an area that, during activation, may be involved in association of the I domain with another region of LFA-1, potentially the -propeller domain. This is the first evidence linking a structural domain of an integrin, in this case the I domain, with a particular activation mechanism.
The Journal of Immunology
The interactions between the leukocyte-specific beta 2-integrins cluster of differentiation (CD) Ag CD11/CD18 and their ligands, the intercellular adhesion molecules (ICAMs), play important roles in many adhesion-dependent leukocyte functions. ICAM-1 is known to be a ligand for both CD11a/CD18 and CD11b/CD18. ICAM-2, whose two extracellular Ig domains show the highest homology to the two NH2-terminal domains of ICAM-1, has been previously shown to be a ligand for CD11a/CD18. We recently found that a 22-amino acid CD11a/CD18-binding peptide, P1, derived from the first domain of ICAM-2, also binds to purified CD11b/CD18. In the present study, we demonstrate that the ICAM-2 protein interacts with CD11b/CD18, and the binding is through the CD11b A domain.
Febs Letters, 1991
The adhesion of human T lymphoblasts to ICAM-l-expressing normal dermal fibroblasts has been assessed as a sensitive model system for the analysis of the interaction of the leucocyte integrin LFA-I with its counter-receptor ICAM-I. Using this model system, the effects of factors known to regulate the activity of LFA-I have been quantitated: temperature; concentration of divalent cations; and exposure to phorbol esters. We show here that under the appropriate assay conditions, this model system represents a useful and simple alternative to the detection of leucocyte binding to purified ICAM-I and also has the additional advantage of permitting more sensitive quantification than is possible using the homotypic adhesion assay.