Joseph K. Welply

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.

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.

Sequencing methods for carbohydrates and their biological applications

Abstract The diversity of oligosaccharide structures may confer the specificity inherent in many molecular and cellular interactions. Scientists from numerous disciplines are faced with the challenge of determining their sequence and deciphering their function.