Susan C. Howard

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.

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‐11–453). Phage bound to immobilized ICAM‐11–453 were eluted by three methods: (1) soluble ICAM‐11–453, (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 EWCEYLGGYLRYCA 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‐11–453 and to ICAM‐11–185, 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‐11–453 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 β2 integrins.

Studies on Selectin-Carbohydrate Interactions

Recruitment of neutrophils to sites of inflammation is now believed to occur through an initial rolling interaction at the luminal surface of activated endothelium and is mediated by a class of mammalian lectins referred to as the selectins. Selectins recognize carbohydrate determinants on co-receptors. It is generally believed that many selectin molecules must bind to many carbohydrate receptor molecules i.e. multivalent binding, to enable sufficient binding strength to elicit the rolling response between the neutrophil and the endothelial cell. One of the approaches to the generation of more potent molecular antagonists of the selectin-mediated cell-cell interaction is to mimic the multivalent interaction in a single compound. Recent experiments utilising conjugated forms of sialyl Lewisx-BSA have explored this feasibility (Welply et al., 1994). In that study, monovalent sLex (sialic acid alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc), the minimum binding determinant for E-selectin, as well as monovalent sialyllactosamine (sialic acid alpha 2-3Gal beta 1-4GlcNAc), a non-binding structure, and the corresponding multivalent BSA-conjugated forms were tested for their ability to inhibit binding of HL-60 cells to immobilised E-selectin. As expected, only sLex and sLex-BSA were found to do so. sLex16-BSA (16 mol tetrasaccharide/mol BSA) showed a dose-dependent inhibition of HL-60 binding with a measured IC50 of 1 microM; demonstrating close to a three-order of magnitude enhancement of inhibitory activity compared to free sLex. This result indicated that multivalent forms of sLex are capable of binding to E-selectin with higher affinity than do monovalent glycans. In another study, fluorescent forms of monovalent sLex were synthesized and used to measure a true thermodynamic dissociation constant for the monovalent sLex:E-selectin interaction of 120 +/- 31 microM (Jacob et.al., 1995).