F₂-isoprostanes are produced in vivo by nonenzymatic peroxidation of arachidonic acid esterified in phospholipids. Increased urinary and plasma F₂-isoprostane levels are associated with a number of human diseases. These metabolites are regarded as excellent markers of oxidant stress in vivo. Isoprostanes are initially generated in situ, i.e. when the arachidonate precursor is esterified in phospholipids, and they are subsequently released in free form. Although the mechanism(s) responsible for the release of free isoprostanes after in situ generation in membrane phospholipids is, for the most part, unknown, this process is likely mediated by phospholipase A₂ activity(ies). Here we reported that human plasma contains an enzymatic activity that catalyzes this reaction. The activity associates with high density and low density lipoprotein and comigrates with platelet-activating factor (PAF) acetylhydrolase on KBr density gradients. Plasma samples from subjects deficient in PAF acetylhydrolase do not release F₂-isoprostanes from esterified precursors. The intracellular PAF acetylhydrolase II, which shares homology to the plasma enzyme, also catalyzes this reaction. We found that both the intracellular and plasma PAF acetylhydrolases have high affinity for esterified F₂-isoprostanes. However, the rate of esterified F₂-isoprostane hydrolysis is much slower compared with the rate of hydrolysis of other substrates utilized by these enzymes. Studies using PAF acetylhydrolase transgenic mice indicated that these animals have a higher capacity to release F₂-isoprostanes compared with nontransgenic littermates. Our results suggested that PAF acetylhydrolases play key roles in the hydrolysis of F₂-isoprostanes esterified on phospholipids in vivo.

Antiviral innate immunity is triggered by sensing viral nucleic acids. RIG-I (retinoic acid-inducible gene-I) is an intracellular molecule that responds to viral nucleic acids and activates downstream signaling, resulting in the induction of members of the type I interferon (IFN) family, which are regarded among the most important effectors of the innate immune system. Although RIG-I is expressed ubiquitously in the cytoplasm, its levels are subject to transcriptional and post-transcriptional regulation. RIG-I belongs to the IFN-stimulated gene (ISG) family, but certain cells regulate its expression through IFN-independent mechanisms. Several lines of evidence indicate that deregulated RIG-I signaling is associated with autoimmune disorders. Further studies suggest that RIG-I has functions in addition to those directly related to its role in RNA sensing and host defense. We have much to learn and discover regarding this interesting cytoplasmic sensor so that we can capitalize on its properties for the treatment of viral infections, immune disorders, cancer, and perhaps other conditions.

The platelet-activating factor acetylhydrolases are enzymes that were initially characterized by their ability to hydrolyze platelet-activating factor (PAF). In human plasma, PAF acetylhydrolase (EC 3.1.1.47) circulates in a complex with low density lipoproteins (LDL) and high density lipoproteins (HDL). This association defines the physical state of PAF acetylhydrolase, confers a long half-life, and is a major determinant of its catalytic efficiency in vivo. The lipoprotein–associated enzyme accounts for all of the PAF hydrolysis in plasma but only two-thirds of the protein mass. To characterize the enzyme–lipoprotein interaction, we employed site-directed mutagenesis techniques. Two domains within the primary sequence of human PAF acetylhydrolase, tyrosine 205 and residues 115 and 116, were important for its binding to LDL. Mutation or deletion of those sequences prevented the association of the enzyme with lipoproteins. When residues 115 and 116 from human PAF acetylhydrolase were introduced into mouse PAF acetylhydrolase (which normally does not associate with LDL), the mutant mouse PAF acetylhydrolase associated with lipoproteins. To analyze the role of apolipoprotein (apo) B100 in the formation of the PAF acetylhydrolase–LDL complex, we tested the ability of PAF acetylhydrolase to bind to lipoproteins containing truncated forms of apoB. These studies indicated that the carboxyl terminus of apoB plays a key role in the association of PAF acetylhydrolase with LDL. These data on the molecular basis of the PAF acetylhydrolase–LDL association provide a new level of understanding regarding the pathway for the catabolism of PAF in human blood.

Asthma, a family of airway disorders characterized by airway inflammation, has an increasing incidence worldwide. Platelet-activating factor (PAF) may play a role in the pathophysiology of asthma. Its proinflammatory actions are antagonized by PAF acetylhydrolase. A missense mutation (V279F) in the PAF acetylhydrolase gene results in the complete loss of activity, which occurs in 4% of the Japanese population. We asked if PAF acetylhydrolase deficiency correlates with the incidence and severity of asthma in Japan. We found that the prevalence of PAF acetylhydrolase deficiency is higher in Japanese asthmatics than healthy subjects and that the severity of this syndrome is highest in homozygous-deficient subjects. We conclude that the PAF acetylhydrolase gene is a modulating locus for the severity of asthma.

The inflammatory response is characterized by a multistep molecular interaction between “signaling” cells, such as endothelial cells, and “responding” cells, such as neutrophils and monocytes. In the first step, selectins produced by signaling cells mediate the tethering of responding cells at sites of inflammation. Subsequently, an additional mediator expressed by signaling cells activates the tethered responding cells. Under pathological conditions, the same mechanism is invoked in inappropriate ways: (1) by prolonged presentation of selectins on the cell surface and (2) by the unregulated production of oxidized phospholipids that mimic the normal secondary signaling molecule, platelet-activating factor (PAF). The enzyme PAF acetylhydrolase (PAF-AH) inactivates PAF and oxidized phospholipids and constitutes an “off” switch that suppresses inflammation. Inhibition of normal PAF-AH function or inactivating mutations of the PAF-AH gene can lead to increased susceptibility to inflammatory disease. These studies have relevance to atherosclerosis and thrombosis, because inflammation is a central feature of both.