High molecular weight homologues of gp91phox, the superoxide-generating subunit of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, have been identified in human (h) and Caenorhabditis elegans (Ce), and are termed Duox for “dual oxidase” because they have both a peroxidase homology domain and a gp91phox domain. A topology model predicts that the enzyme will utilize cytosolic NADPH to generate reactive oxygen, but the function of the ecto peroxidase domain was unknown. Ce-Duox1 is expressed in hypodermal cells underlying the cuticle of larval animals. To investigate function, RNA interference (RNAi) was carried out in C. elegans. RNAi animals showed complex phenotypes similar to those described previously in mutations in collagen biosynthesis that are known to affect the cuticle, an extracellular matrix. Electron micrographs showed gross abnormalities in the cuticle of RNAi animals. In cuticle, collagen and other proteins are cross-linked via di- and trityrosine linkages, and these linkages were absent in RNAi animals. The expressed peroxidase domains of both Ce-Duox1 and h-Duox showed peroxidase activity and catalyzed cross-linking of free tyrosine ethyl ester. Thus, Ce-Duox catalyzes the cross-linking of tyrosine residues involved in the stabilization of cuticular extracellular matrix.

Active migration of polymorphonuclear leukocytes (PMN) through the intestinal crypt epithelium is a hallmark of inflammatory bowel disease and correlates with patient symptoms. Previous in vitro studies have shown that PMN transepithelial migration results in increased epithelial permeability. In this study, we modeled PMN transepithelial migration across T84 monolayers and demonstrated that enhanced paracellular permeability to small solutes occurred in the absence of transepithelial migration but required both PMN contact with the epithelial cell basolateral membrane and a transepithelial chemotactic gradient. Early events that occurred before PMN entering the paracellular space included increased permeability to small solutes (<500 Da), enhanced phosphorylation of regulatory myosin L chain, and other as yet undefined proteins at the level of the tight junction. No redistribution or loss of tight junction proteins was detected in these monolayers. Late events, occurring during actual PMN transepithelial migration, included redistribution of epithelial serine-phosphorylated proteins from the cytoplasm to the nucleus in cells adjacent to migrating PMN. Changes in phosphorylation of multiple proteins were observed in whole cell lysates prepared from PMN-stimulated epithelial cells. We propose that regulation of PMN transepithelial migration is mediated, in part, by sequential signaling events between migrating PMN and the epithelium.

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The integrin CD11c/CD18 plays a role in leukocyte and cell matrix adhesion and is highly expressed in certain hematopoietic malignancies. To better characterize ligand binding properties, we panned random peptide phage‐display libraries over purified CD11c/CD18. We identified a phage expressing the circular peptide C‐GRWSGWPADL‐C. C‐GRWSGWPADL‐C phage bound specifically to CD11c/CD18 expressing monocytes but not CD11c/CD18 negative lymphocytes and showed 5 × 103‐fold higher binding to purified CD11c/CD18 than control phage, without binding to CD11b/CD18. Peptide sequence analysis revealed a similar sequence in domain D5 of ICAM‐1 and an alternative, phase‐shifted motif in domain D4. Surface plasmon resonance experiments demonstrated direct interaction of ICAM‐1 and CD11c/CD18. A soluble fusion protein containing the extracellular domain of ICAM‐1 abolished C‐GRWSGWPADL‐C phage binding to CD11c/CD18. Moreover, synthetic monomeric circular peptide C‐GRWSGWPADL‐C bound specifically to CD11c/CD18 and inhibited ICAM‐1 binding. Its rather low binding affinity and inability to displace pentavalent C‐GRWSGWPADL‐C phage from CD11c/CD18 suggests that a multimeric display of the selected peptide is essential for high affinity binding. Using ICAM‐1 deletion constructs, we showed that domain D4 is required for interaction with CD11c/CD18, suggesting that C‐GRWSGWPADL‐C phage binds specifically to CD11c/CD18 by structurally mimicking the interaction site on D4 of ICAM‐1.

Infiltration and accumulation of polymorphonuclear leukocytes within the tissues is a hallmark of the acute inflammatory response. A prominent feature of acute inflammation is enhanced vascular permeability resulting in edema formation. Such changes in vascular permeability have been known to be dependent upon polymorphonuclear leukocyte interactions with the vascular endothelium. Careful investigation has shown clearly that permeability changes can occur without polymorphonuclear leukocyte transendothelial migration, and that polymorphonuclear leukocyte migration can occur without permeability alteration. The underlying mechanisms of polymorphonuclear leukocyte-stimulated changes in endothelial barrier function have remained elusive. Endothelial activation and polymorphonuclear leukocyte adhesion to the endothelium are both required for polymorphonuclear leukocyte-induced changes in vascular permeability. Polymorphonuclear leukocyte-derived azurocidin plays a major role in this polymorphonuclear leukocyte-evoked alteration in endothelial permeability. Azurocidin is released after activation of polymorphonuclear leukocytes, such as after ligation of the major adhesive integrin CD11b/CD18. Understanding how polymorphonuclear leukocytes alter vascular permeability may provide targets for new drugs for appropriate therapeutic intervention in acute and chronic inflammatory diseases. This review focuses on the role of polymorphonuclear leukocyte-derived azurocidin in alteration of vascular permeability.