T. Imaizumi

Platelet-activating factor acetylhydrolase deficiency. A missense mutation near the active site of an anti-inflammatory phospholipase.

Deficiency of plasma platelet-activating factor (PAF) acetylhydrolase is an autosomal recessive syndrome that has been associated with severe asthma in Japanese children. Acquired deficiency has been described in several human diseases usually associated with severe inflammation. PAF acetylhydrolase catalyzes the degradation of PAF and related phospholipids, which have proinflammatory, allergic, and prothrombotic properties. Thus, a deficiency in the degradation of these lipids should increase the susceptibility to inflammatory and allergic disorders. Miwa et al. reported that PAF acetylhydrolase activity is absent in 4% of the Japanese population, which suggests that it could be a common factor in such disorders, but the molecular basis of the defect is unknown. We show that inherited deficiency of PAF acetylhydrolase is the result of a point mutation in exon 9 and that this mutation completely abolishes enzymatic activity. This mutation is the cause of the lack of enzymatic activity as expression in E. coli of a construct harboring the mutation results in an inactive protein. This mutation as a heterozygous trait is present in 27% in the Japanese population. This finding will allow rapid identification of subjects predisposed to severe asthma and other PAF-mediated disorders.

Platelet-activating factor (PAF) stimulates the production of PAF acetylhydrolase by the human hepatoma cell line, HepG2.

The human hepatoma cell line, HepG2, secreted an activity that degrades platelet-activating factor (PAF) by the hydrolysis of the sn-2 acetyl group. This activity was Ca++ independent, inhibited by diisopropylfluorophosphate but not by p-bromophenacyl bromide, and resistant to treatment with trypsin or pronase. Separation of HepG2-conditioned medium by gel filtration disclosed that the activity was associated with lipoproteins. An antiserum against PAF acetylhydrolase immunoprecipitated this activity. It was not recognized by an antibody against lecithin:cholesterol acyltransferase (LCAT), which also is secreted by HepG2 cells. Therefore the phospholipase A2 activity of LCAT was excluded as a source of the observed activity. PAF added to the culture medium stimulated the secretion of the PAF-degrading activity by HepG2 cells, while lyso-PAF was inactive. Maximal stimulation was observed with 5 ng/ml PAF, which induced a fivefold increase. The presence of 5 ng/ml PAF, enhanced the secretion of [35S]methionine-labeled PAF acetylhydrolase and cycloheximide inhibited both the basal and PAF-stimulated secretion of the labeled enzyme. We conclude that HepG2 cells produce PAF acetylhydrolase. The liver may be a major source of plasma PAF acetylhydrolase, and PAF may induce the production of its inactivating enzyme by the liver.

Increased levels of blood platelet-activating factor (PAF) and PAF-like lipids in patients with ischemic stroke.

Levels of platelet‐activating factor (PAF) in blood from patients with ischemic stroke were determined by radioimmunoassay (RIA). Using 2 ml of blood as a starting material, PAF was detected in 11 out of 17 stroke patients and 3 of 25 age‐matched healthy controls. This implies that blood level of PAF is higher in stroke patients than in controls. Plasma levels of PAF‐like lipid(s) (PAF‐LL) were also estimated in the same subjects by a bioassay based on aggregation of human polymorphonuclear neutrophils. PAF‐LL was detected in plasma samples of all subjects and the average values in patients and controls were 294 ± 211 pg/ml and 140 ± 122 pg/ml, respectively. There was a statistically significant difference between these two values (p < 0.01). Separation of plasma lipids by HPLC gave a single peak in bioassay, which had the same elution volume as authentic PAF. When each fraction was subjected to RIA, the fractions corresponded to phosphatidylcholine (PC) or lysoPC also showed the immunoreactivity, however, the purification procedure using an octadecylsilica gel cartridge eliminated such cross‐reacting compounds. We conclude that blood PAF is higher in patients with ischemic stroke than in healthy subjects. Besides, there may be bioactive phospholipid molecules other than PAF, which level in plasma is also higher in stroke patients.

Cisplatin inhibits the expression of X-chromosome-linked inhibitor of apoptosis protein in an oral carcinoma cell line

Cisplatin is known to activate caspase-3 through the mitogen-activated kinase (MAPK) pathway. We found that X-chromosome-linked inhibitor of apoptosis protein (XIAP), a direct inhibitor of caspase-3, 7, and 9, is constitutively expressed in a cell line of oral squamous cell carcinoma, KOSC-2. Cisplatin treatment of the cells inhibited the expression of XIAP, and this was associated with DNA fragmentation. Overexpression of XIAP, by a transfection experiment, inhibited the cisplatin-induced apoptosis. We conclude that cisplatin induces apoptosis mediated not only by the activation of MAPK pathway but by the inhibition of XIAP.

Bacterial lipopolysaccharide induces endothelial cells to synthesize a degranulating factor for neutrophils

Enzymes and other factors secreted by degranulating neutrophils (polymorphonuclear leukocytes, PMNs) mediate endothelial injury, thrombosis, and vascular remodeling. In bacteremia and sepsis syndrome and their consequent complications (including acute respiratory distress syndrome and systemic ischemia‐reperfusion resulting from septic shock), neutrophil degranulation is an important mechanism of injury. In related studies, we found that human endothelial cells regulate neutrophil degranulation and that inflammatory cytokines induce synthesis of degranulating factors by human endothelial cells. Here we show that lipopolysaccharides (LPS) from gram‐negative bacteria were the most potent agonists for release of degranulating activity by endothelial cells when compared to several cytokines and stimulatory factors. LPS also induced the release of degranulating signals for PMNs from a human endothelial cell line, EA.hy 926. Interleukin 8 (IL‐8) is synthesized by endothelial and EA.hy 926 cells in response to LPS and induces neutrophil degranulation. However, complementary strategies using receptor desensitization, translation of messenger RNA by Xenopus laevis oocytes, and purification and analysis of factors from conditioned supernatants demonstrated that degranulating factors distinct from IL‐8 are generated in response to LPS. The characteristics of a partially purified degranulating factor isolated from conditioned supernatants distinguished it from known chemokines and other factors that induce PMN degranulation and are generated by endothelial cells in response to LPS. Thus, cultured human endothelial cells and endothelial cell lines synthesize several unique signaling molecules that can trigger neutrophil granular secretion. If produced in vivo in response to LPS or other pathologic agonists, these degranulating signals may activate PMNs in combination or in sequence, initiating or propagating vascular damage.—Gill, E. A., Imaizumi, T.‐a., Carveth, H., Topham, M. K., Tarbet, E. B., Mcintyre, T. M., Prescott, S. M., Zimmerman, G. A. Bacterial lipopolysaccharide induces endothelial cells to synthesize a degranulating factor for neutrophils. FASEB J. 12, 673–684 (1998)

Platelet‐activating factor acetylhydrolase activity in red blood cell‐stroma from patients with cerebral thrombosis

Platelet‐activating factor (1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine, PAF) is a bioactive phospholipid and inactivated by a specific enzyme, PAF acetylhydrolase (PAF‐AH). We have measured PAF‐AH activity in red blood cells (RBCs) from patients with a history of cerebral thrombosis and age‐matched healthy controls. The activities in 34 patients and 34 controls were 1.29 ± 0.28 and 1.64 ± 0.26 nmol/109 RBCs/min (or, 30.0 ± 5.8 and 35.1 ± 4.7 nmol/g protein/min) (mean ± SD), respectively, and the difference was significant (p < 0.001). In patients, RBC PAF‐AH activity correlated positively with RBC filterability, an index of RBC deformability (r = +0.501, p < 0.05). The RBC activity may play a role in scavenging oxidation products of membrane phospholipids. Lower RBC PAF‐AH activity may predispose to ischemic diseases by disturbing microcirculatory behavior of the RBCs.

Plasma 11-dehydrothromboxane B2: a reliable indicator of platelet hyperfunction in patients with ischemic stroke

The plasma level of 11‐dehydrothromboxane B2 (11‐dehydroTXB2) is free from artifactual increase during blood sampling, and it can be a reliable indicator of TXA2 production in vivo. We have estimated plasma 11‐dehydroTXB2 in patients with ischemic stroke. Subjects studied were 29 patients with cerebral thrombosis (62 ± 9 years old) and 41 healthy controls (61 ± 1 years old). Plasma 11‐dehydroTXB2 and TXB2 were determined by radioimmunoassay. Plasma 11‐dehydroTXB2 levels in patients and controls were 5.4 ± 2.5 and 1.8 ± 0.9 pg/ml, respectively, and the difference was significant (p < 0.001). Plasma TXB2 also was higher in patients than in controls: 401 ± 61 vs 311 ± 51 pg/ml (p < 0.05). However, the 11‐dehydroTXB2 was found to be a more effective parameter to distinguish between stroke patients and controls. Estimation of plasma 11‐dehydroTXB2 levels is a reliable method to detect platelet hyperfunction in stroke patients.

Density-associated changes in platelet-activating factor acetylhydrolase activity and membrane fluidity of human erythrocytes

SummaryPlatelet-activating factor acetylhydrolase is known to degrade oxidatively fragmented phospholipids which are similar in structure to platelet-activating factor. We examined changes of acetylhydrolase activity during in vivo aging of human erythrocytes and tried to assess its role in maintaining the membrane properties of erythrocytes. Higher-density erythrocytes are enriched with older cells. Erythrocytes obtained from seven healthy colleagues were separated into four density fractions by centrifugation in discontinuous Percoll density gradients. Both membrane and cytosolic acetylhydrolase decreased with increasing erythrocyte density. Membrane and cytosolic acetylhydrolase activities in the lightest fraction were 2.0±1.0 (SD) nkat/g protein and 362±58 pkat/g protein, respectivley, and these values were significantly higher than those in the densest fraction: 1.3±0.7 nkat/g protein and 286±70 pkat/g protein, respectively. Membrane acyltransferase activity also decreased with red cell density and the average values in the lightest and densest fractions were 51.2±23.6 and 27.0±20.2 μkat/g protein, respectively. Generation of thiobarbituric acid-reactive substances induced byt-butyl hydroperoxide treatment decreased with increasing cell density, and the inhibition of acetylhydrolase with diisopropylfluorophosphate resulted in enhanced peroxide-induced lipid oxidation, particularly in lower-density fractions. There was no significant change in basal levels of thiobarbituric acid-reactive substances in red cell membrane. Membrane fluidity was evaluated by fluorescence recovery after photobleaching and it decreased as erythrocyte density increased. We conclude that the activity of the deacylation/reacylation cycle maintained by acetylhydrolase and acyltransferase is gradually reduced during in vivo aging of erythrocytes. This may be connected with decreases of polyunsaturated fatty acids and membrane fluidity in old eryhtrocytes.

An oxidized derivative of cholesterol increases the release of soluble vascular cell adhesion molecule-1 from human umbilical vein endotherial cells in culture

Treatment of human umbilical vein endothelial cells (HUVECs) with 7-ketocholesterol resulted in an increased release of soluble vascular cell adhesion molecule-1 (VCAM-1) into culture medium. 7-Ketocholesterol did not enhance the expression of mRNA for VCAM-1. 7 beta-Hydroxy- or 25-hydroxycholesterol had no effect on soluble VCAM-1 levels. Western blot analysis revealed that soluble VCAM-1, in the conditioned medium of both 7-ketocholesterol-stimulated and control cells, had a molecular size of 100 kDa. Stimulation of the TNF-alpha-treated HUVECs with 7-ketocholesterol further increased the levels of soluble VCAM-1 in the culture medium. Again, 7-ketocholesterol did not affect the VCAM-1 mRNA level, which was enhanced by TNF-alpha. Pretreatment of the cells with tissue inhibitor of membrane metalloproteinase-2 (TIMP-2) completely inhibited the release of VCAM-1 in response to 7-ketocholesterol but TIMP-1 had no effect. Adherence of mononuclear cells to TNF-stimulated HUVEC monolayers was slightly inhibited by 7-ketocholesterol, but this oxysterol did not affect the basal adherence to non-stimulated HUVECs. Immunofluorescent staining of the cells confirmed diffuse perinuclear distribution of VCAM-1 in HUVECs treated with TNF-alpha, but 7-ketocholesterol did not affect the intensity or distribution of immunofluorescence. We conclude that 7-ketocholesterol releases VCAM-1 from the endothelium probably by a proteolytic process.