Tissue factor and factor VIIa receptor/ligand interactions induce proinflammatory effects in macrophages.

The potential for tissue factor (TF) to enhance inflammation by factor VIIa-dependent induction of proinflammatory changes in macrophages was explored. Purified recombinant human factor VIIa enhanced reactive oxygen species production by human monocyte-derived macrophages expressing TF in vitro. This effect was dose- and time-dependent, ligand- and receptor-specific, and independent of other coagulation proteins. This receptor/ligand binding induced phospholipase C-dependent intracellular calcium fluxes. Transfection studies using a human monocyte-derived cell line (U937) demonstrated that an intact intracytoplasmic domain of TF is required for factor VIIa-induced intracellular calcium fluxes. The capacity of TF to enhance proinflammatory functions of rabbit peritoneal-elicited macrophages (production of reactive oxygen species and expression of major histocompatibility complex class II and cell adhesion molecules) was demonstrated in vivo by treatment with an anti-TF antibody. These data demonstrate that, in addition to its role in activation of coagulation, TF can directly augment macrophage activation. These effects are initiated by binding factor VIIa and are independent of other coagulation proteins. These studies provide the first demonstration of a direct proinflammatory role for TF acting as a cell-signaling receptor.

4-Methylphthalate catabolism in Burkholderia (Pseudomonas) cepacia Pc701: a gene encoding a phthalate-specific permease forms part of a novel gene cluster.

We have determined the entire nucleotide sequence of a 4.4 kbp fragment of pMOP, a plasmid involved in 4-methylphthalate catabolism in Burkholderia cepacia (formerly Pseudomonas cepacia) Pc701. Two complete ORFs were identified and termed mopA and mopB. mopB encodes a 4-methylphthalate permease which is a member of a superfamily of symport proteins found in both prokaryotes and eukaryotes. Functionality was assigned to MopB by detailed analysis of the predicted amino acid sequence, resulting in the identification of 12 hydrophobic membrane-spanning domains and motifs associated with this class of protein. An assay was developed to demonstrate MopB function in substrate uptake. Of 4-methylphthalate, 4-hydroxyisophthalate, benzoate, p-toluate and phthalate, only uptake of 4-methylphthalate and phthalate was demonstrated, suggesting that two carboxyl groups in the ortho position are essential for substrate recognition. The predicted protein MopA showed significant levels of homology to reductase proteins implicated in aromatic and aliphatic catabolism, and contained motifs recognized as binding the ADP and flavin moieties of FAD/NAD. Northern hybridization experiments determined that mopA and mopB are contranscribed, but expression was only seen in cells grown on 4-methylphthalate and not in cells grown on closely related structural analogues, including phthalate. mopA and mopB may be situated at the 3' terminus of a cistron about 10 kbp in size. The isolation and characterization of a 4-methylphthalate permease gene may lead to the identification of other permeases involved in bacterial biodegradation processes and possibly the construction of strains with enhanced degradative abilities.