A peptide isolated by phage display binds to ICAM-1 and inhibits binding to LFA-1.

A mixed phage library containing random peptides from four to eight residues in length flanked by cysteine residues was screened using a recombinant soluble, form of human ICAM-1, which included residues 1-453, (ICAM-1(1-453)). Phage bound to immobilized ICAM-1(1-453) were eluted by three methods: (1) soluble ICAM-1(1-453), (2) neutralizing murine monoclonal antibody, (anti-ICAM-1, M174F5B7), (3) acidic conditions. After three rounds of binding and elution, a single, unique ICAM-1 binding phage bearing the peptide EWCEYLGGYLRCYA was isolated; the identical phage was selected with each method of elution. Attempts to isolate phage from non-constrained (i.e., not containing cysteines) libraries did not yield a phage that bound to ICAM-1. Phage displaying EWCEYLGGYLRCYA bound to immobilized ICAM-1(1-453) and to ICAM-1(1-185), a recombinant ICAM-1, which contains only the two amino-terminal immunoglobulin domains residing within residues 1-185. This is the region of the ICAM-1 that is bound by LFA-1. The phage did not bind to proteins other than ICAM-1. The phage bound to two ICAM-1 mutants, which contained amino acid substitutions that dramatically decreased or eliminated the binding to LFA-1. Studies were also performed with the corresponding synthetic peptide. The linear form of the synthetic EWCEYLGGYLRCYA peptide was found to inhibit LFA-1 binding to immobilized ICAM-1(1-453) in a protein-protein binding assay. By contrast, the disulfide, cyclized, form of the peptide was inactive. The EWCEYL portion of the sequence is homologous to the EWPEYL sequence found within rhinovirus coat protein 14, a nonintegrin protein that binds to ICAM-1. Taken together, the results suggests that the EWCEYLGGYLRCYA sequence is capable to binding to immobilized ICAM-1. Phage display appears to represent a new approach for the identification of peptides that interfere with ICAM-1 binding to beta 2 integrins.

Binding of sialyl Lewis x to E-selectin as measured by fluorescence polarization.

Fluorescence polarization has been used to directly measure the binding of the tetrasaccharide sialyl Lewisx (sLe(x)[Glc], or NeuAc alpha 2-3Gal beta 1-4[Fuc alpha 1-3]Glc) to a soluble form of E-selectin, a member of the class of adhesion molecules that plays an important role in immune-cell response to inflammation. The experiments utilized a fluorescent derivative of sLe(x)[Glc] with fluorescein attached directly to the glucose residue through a beta-glycosidic linkage. The resulting fluorescent sLe(x) was shown to inhibit binding of HL60 cells to immobilized E-selectin and exhibited fluorescence polarization enhancement in the presence of a monovalent form of a recombinant soluble E-selectin-Fc chimera. Thermodynamic dissociation constants of 107 +/- 26 and 120 +/- 31 microM were obtained for the fluorescent sLe(x)[Glc] and the free sLe(x)[Glc] sugars, respectively. These results demonstrate that E-selectin interacts weakly with the minimal carbohydrate recognition determinant sLe(x). Additional binding interactions through the action of the authentic coreceptor or via clustering of the ligand and E-selectin molecules on the respective neutrophil and endothelial cell surfaces may also play a role in the overall cellular binding strength. However, the basic interaction between carbohydrate and protein appears weak, consistent with other carbohydrate-protein interactions studied to date.

Intratracheal administration of endotoxin and cytokines: VIII. LPS induces E-selectin expression; anti-E-selectin and soluble E-selectin inhibit acute inflammation.

E-selectin is an inducible endothelial adhesion molecule that binds neutrophils. E-selectin mRNA is not constitutively detectable in the lungs of rats. Intratracheal injection of LPS induces pulmonary E-selectin mRNA expression at 2-4 h. Intratracheal injection of LPS followed at 2 and 4 h by intravenous injection of mouse F(ab')2 or F(ab') anti-E-selectin monoclonal antibody inhibits the emigration of neutrophils into the bronchoalveolar space at 6 h by 50-70%. TNF and IL-6 bioactivity are not decreased in bronchoalveolar lavage fluid after treatment with anti-E-selectin antibody as compared to controls, suggesting that the anti-E-selectin does not affect the magnitude of the LPS-initiated cytokine cascade. Intratracheal injection of LPS followed at 2 and 4 h by intravenous injection of soluble E-selectin inhibits neutrophilic emigration at 6 h by 64%, suggesting that endogenous soluble E-selectin shed from activated endothelium may play a role in the endogenous down-regulation of acute inflammation. E-selectin-mediated adhesion of neutrophils to endothelium appears crucial to the full development of the acute inflammation response.

The glycoprotease of Pasteurella haemolytica A1 eliminates binding of myeloid cells to P-selectin but not to E-selectin.

HL-60 cells and neutrophils treated with the glycoprotease from Pasteurella haemolytica A1, an enzyme which is specific for O-sialoglycoproteins, were found to be incapable of binding P-selectin but still bound E-selectin. Comparative analysis of [35-S] cysteine labeled proteins from HL-60 cells by 2-dimensional electrophoresis indicated that two major proteins with M(r) 100 and 115 kd were significantly removed from cells which had been treated.