Tyrosine cross-linking of extracellular matrix is catalyzed by Duox, a multidomain oxidase/peroxidase with homology to the phagocyte oxidase subunit gp91phox.

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.

Novel G protein-coupled responses in leukocytes elicited by a chemotactic bacteriophage displaying a cell type-selective binding peptide.

Recently, we identified a neutrophil-binding phage displaying a novel peptide motif, GPNLTGRW. It was determined that this peptide, when displayed on bacteriophage (FGP phage), elicits a transient increase in cytosolic calcium. Here, we show that FGP phage stimulate neutrophil chemotaxis and induce a pertussis toxin-sensitive rise in cytosolic calcium in monocytes as well as in neutrophils. In contrast to the calcium response elicited by classical chemoattractants fMLP and IL-8, the FGP phage-elicited response in neutrophils is dependent on extracellular calcium and is mediated by receptor-activated, divalent cation channels. Consistent with G protein-coupled receptor signaling, FGP phage effect homologous and reciprocal heterologous desensitization with fMLP- and IL-8-stimulated calcium responses. Like non-G protein-coupled responses, the FGP-elicited calcium transient is abolished with phosphoinositide-3-kinase inactivation. Nonetheless, specific binding of GTP to neutrophil membranes follows stimulation with FGP phage, further supporting involvement of G proteins. However, FGP phage neither bind to nor elicit a calcium response from transfectant cells harboring known candidate G protein-coupled receptors. These data together suggest that the elicited responses are mediated by a novel G protein-coupled receptor or represent novel responses of a known receptor.

Modulation of epithelial and endothelial paracellular permeability by leukocytes.

The tight junction acts as a regulated barrier for diffusion of ions, larger solutes, and migration of leukocytes through the paracellular space. Barrier function varies with the tissue type, and positively correlates with the number and complexity of tight junction strand formation. Polarized epithelia and brain capillary endothelium display a high degree of barrier function and tight junction formation. Conversely, vascular endothelium in other tissues has fewer, loosely organized tight junctions strands and greater permeability. Through cytoskeletal association, the tight junction and the adherens junction form a functional unit termed the apical junction complex. The development and stabilization of the tight junction is dependent upon the adherens junction. It has become apparent that paracellular permeability is altered during the migration of leukocytes across the apical junction complex and that the apical junction complex plays an important role in the regulation of leukocyte transmigration (or extravasation) through the endothelium and epithelium. Evidence suggests that important cell-cell adhesive events between transmigrating leukocytes and the apical junction complex and subsequent signaling events result in the facilitation of the passage of cells through the paracellular space. Possible mechanisms for the regulation of barrier function pertaining to leukocyte transmigration are discussed.

Non-serum-dependent chemotactic factors produced by Candida albicans stimulate chemotaxis by binding to the formyl peptide receptor on neutrophils and to an unknown receptor on macrophages.

Serum-free culture filtrates of six Candida species and Saccharomyces cerevisiae were found to contain chemoattractants for human polymorphonuclear leukocytes (PMNs) and a mouse macrophage-like cell line, J774. The chemotactic factors differed for the PMN and J774 cells, however, in terms of heat stability, kinetics of liberation by the yeast cells, and divalent cation requirements for production. The chemoattractant in Candida albicans culture filtrates appeared to act through the formyl peptide receptor (FPR) of PMNs, since it was found to induce chemotaxis of Chinese hamster ovary (CHO) cells that were expressing the human FPR but did not induce chemotaxis of wild-type CHO cells. The C. albicans culture filtrates also induced migration of PMNs across confluent monolayers of a human gastrointestinal epithelial cell line, T84; migration occurred in the basolateral-to-apical direction but not the reverse direction, unless the epithelial tight junctions were disrupted. J774 cells did not migrate toward the formylated peptide (fMet-Leu-Phe; fMLF), and chemotaxis toward the C. albicans culture filtrate was not inhibited by an FPR antagonist (t-butoxycarbonyl-Met-Leu-Phe), suggesting that a different receptor mediated J774 cell chemotaxis. In conclusion, we have identified a receptor by which a non-serum-dependent chemotactic factor (NSCF) produced by C. albicans induced chemotaxis of PMNs. Additionally, we have shown that NSCF was active across epithelial monolayers. These findings suggest that NSCFs produced by C. albicans and other yeast species may influence host-pathogen interactions at the gastrointestinal tract mucosal surface by inducing phagocytic-cell infiltration.

Analysis of surface antigen expression and host defense function in leukocytes from calves heterozygous or homozygous for bovine leukocyte adhesion deficiency.

OBJECTIVE: To analyze surface antigen expression and functional responses of leukocytes from calves heterozygous and homozygous for bovine leukocyte adhesion deficiency (BLAD). ANIMALS: 8 clinically normal calves, 4 calves heterozygous for BLAD, and 4 calves homozygous for BLAD. PROCEDURE: Surface antigen expression was examined by flow cytometric analysis of leukocytes stained with monoclonal antibodies. Neutrophil function analyses included phagocytosis and killing of Candida albicans and measurement of respiratory burst activity using cytochrome c and dihydrorhodamine 123 assays. Differential leukocyte counts also were performed. RESULTS: Leukocytes from heterozygous calves were similar to those of clinically normal calves with respect to surface antigen expression, C albicans phagocytosis and killing, and respiratory burst activity. In contrast, neutrophils from calves homozygous for BLAD had significantly reduced phagocytic and yeast-killing capacity but had higher respiratory burst activity than cells from clinically normal or heterozygous calves. Homozygous calves also had extreme neutrophilia and significantly more immature neutrophils. CONCLUSIONS: The heterozygous BLAD genotype does not cause detectable functional differences in leukocytes, compared with those of clinically normal calves. In contrast, leukocytes from homozygous calves seem to upregulate alternative host defense capabilities (eg, respiratory burst activity) to partially compensate for the lack of typical adherence-dependent host defense functions.