Reversible membrane association of neutrophil 5-lipoxygenase is accompanied by retention of activity and a change in substrate specificity.

Ionophore activation of the human polymorphonuclear neutrophil results in eicosanoid synthesis and the accumulation of inactive 5-lipoxygenase in a membrane compartment. We report here that inhibition of self-inactivation of 5-lipoxygenase in ionophore-treated neutrophils with the reversible inhibitor zileuton, results in the accumulation of active 5-lipoxygenase in the membrane fraction. In zileuton plus ionophore-treated cells, 77% of the specific activity of the cytosolic enzyme from resting cells was diverted to the membrane fraction compared to 22% of the activity translocated when ionophore alone was used to activate the neutrophils. Accumulation of active membrane-associated 5-lipoxygenase was inhibited and reversed by the 5-lipoxygenase translocation inhibitor MK-886. The membrane-associated 5-lipoxygenase was two times more efficient in the production of leukotriene A4 from arachidonate-derived 5-hydroperoxyeicosatetraenoic acid than the cytosolic enzyme. Unlike the cytosolic enzyme, membrane-associated 5-lipoxygenase could metabolize 12(S)- and 15(S)-hydroxyeicosatetraenoic acid to 5(S),12(S)- and 5(S),15(S)-dihydroxyeicosatetraenoic acid, respectively. The ability to metabolize hydroxy fatty acids was dependent upon 5-lipoxygenase-activating protein association, but was lost if 5-lipoxygenase was eluted from the membrane by MK-886. These studies reveal for the first time that significant quantities of active 5-lipoxygenase can be detected in the membrane fraction of activated neutrophils and show that membrane association can alter the substrate specificity of 5-lipoxygenase which is further evidence for the role of the membrane-associated enzyme in the synthesis of 5-lipoxygenase metabolites.     

Stimulatory effect of menaquinone-7 (vitamim K2) on osteoblastic bone formation in vitro

Menaquinone-7, which is vitamin K₂ (menatetrenone) with seven isoprene units, is highly contained in the fermented soybean. The effect of menaquinone-7 (MK-7) on osteoblastic bone formation was investigated. Femoral-diaphyseal and metaphyseal tissues of young male rats (4 weeks old) were cultured for 48 h in a medium containing either vehicle or MK-7 (10^–7–10^–5 M). Calcium content, alkaline phosphatase activity, and deoxyribonuclic acid (DNA) content in the diaphyseal and metaphyseal tissues was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing the diaphyseal and metaphyseal calcium content and alkaline phosphatase activity was completely prevented in the presence of cycloheximide (10^–6 M), an inhibitor of protein synthesis. Moreover, osteoblastic MC3T3-E1 cells after subculture were cultured for 24 h in a serum-free medium containing MK-7 (10^–7–10^–5 M). Protein content, alkaline phophatase activity, osteocalcin and DNA content in the cells was significantly increased in the presence of MK-7 (10^–6 and 10^–5 M). The effect of MK-7 in increasing protein content, alkaline phosphatase activity, and osteocalcin production in the cells was completely blocked by cycloheximide. This study demonstrates that MK-7 has an anabolic effect on bone tissue and osteoblastic MC3T3-E1 cells in vitro, suggesting that the compound can stimulate osteoblastic bone formation.     

Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y2 receptor

Nucleotides are released during vascular injury from activated platelets and broken cells, which could stimulate human neutrophils. In this study, we characterized the P2Y receptors and investigated the functional effects of extracellular nucleotides on human neutrophils. Pharmacological characterization using selective agonists and pertussis toxin revealed that human neutrophils express only functional P2Y₂ receptors. However, P2Y₂ receptor agonists ATP or uridine triphosphate (UTP) caused intracellular Ca²⁺ increases in isolated human neutrophils with an EC₅₀ of 1 µM but failed to cause release of primary granules from human neutrophils. ATP and UTP were equally potent in causing elastase release from human neutrophils in the presence of exogenous soluble fibrinogen, whereas ADP and UDP were without effect. We investigated whether nucleotides depend on generated arachidonic acid metabolites to cause degranulation. However, phenidone and MK-886, inhibitors of the 5-lipoxygenase pathway, failed to block nucleotide-induced intracellular calcium mobilization and elastase release. ATP and UTP caused activation of p38 MAPK and ERK1/2 in human neutrophils. In addition, the inhibitors of the MAPK pathway, SB-203580 and U-0126, inhibited nucleotide-induced elastase release. We conclude that fibrinogen is required for nucleotide-induced primary granule release from human neutrophils through the P2Y₂ receptor without a role for arachidonic acid metabolites. Both ERK1/2 and p38 MAPK play an important role in nucleotide-induced primary granule release from human neutrophils. elastase release; fibrinogen; extracellular nucleotides; uridine triphosphate; adenosine triphosphate     

Control of experimental pulmonary tuberculosis depends more on immunostimulatory leukotrienes than on the absence of immunosuppressive prostaglandins

Prostaglandins (PGs) and leukotrienes (LTs) are produced in Mycobacterium tuberculosis (Mtb)-infected lungs and have immune suppressive and protective effects, respectively. Considering that both of these mediators are produced during mycobacterial infection, we investigated the specific and relative biological importance of each in regulating host response in experimental tuberculosis. Administration of celecoxib, which was found to reduce lung levels of PGE₂ and increase LTB₄, enhanced the 60-day survival of Mtb-infected mice in 14%. However administration of MK-886, which reduced levels of LTB₄ but did not enhance PGE₂, reduced 60-day survival from 86% to 43% in Mtb-infected mice, and increased lung bacterial burden. MK-886 plus celecoxib reduced survival to a lesser extent than MK-886 alone. MK-886- and MK-886 plus celecoxib-treated animals exhibited reduced levels of the protective interleukin-12 and gamma-interferon. Our findings indicate that in this model, the protective effect of LTs dominates over the suppressive effect of PGs.     

Nitric oxide mediates distinct effects of various LPS chemotypes on phagocytosis and leukotriene synthesis in human neutrophils

We investigated the effect of lipopolysaccharide (LPS) chemotypes differing in their carbohydrate chain length on phagocytosis of serum-opsonized zymosan (OZ) particles and related functions of human polymorphonuclear leukocyte (PMNL, neutrophils). LPS from deep core mutant (Re), complete core (Ra) and smooth (S) phenotypes of Salmonella typhimurium was studied. Priming of neutrophils with various LPSs caused prominent enhancement of OZ phagocytosis, superoxide production and leukotriene (LT) synthesis in neutrophils, with LPS effects increasing as Re < S < Ra. The LPS forms were less potent to activate OZ uptake in the presence of MK-886, 5-lipoxygenase activating protein inhibitor, suggesting the regulatory function of 5-lipoxygenase (5-LO)-derived LTs. Direct measurement of nitrite release from OZ-stimulated neutrophils revealed that the effects of LPS on NO synthesis increased in the range of Ra < S < Re. Nitric oxide synthase (NOS) inhibitor l-NAME increased phagocytosis, LT and superoxide formation by neutrophils, and abolished the difference in the action of the LPSs forms. Further mechanistic studies revealed that NO modulates cellular 5-LO activity in a guanylyl cyclase and protein kinase G dependent manner, as well as interplay between NO and superoxide, and peroxynitrite generation contribute to distinct effects of LPS chemotypes on phagocytosis and LT synthesis in human neutrophils. Our investigation of the three LPS species demonstrates that the LPS polysaccharide core is mostly essential for the PMNL activation and is able to suppress lipid A-induced increase in NOS activity in phagocyting cells by triggering specific signaling cascades.     

IL-18 enhances collagen-induced arthritis by recruiting neutrophils via TNF-alpha and leukotriene B4

IL-18 expression and functional activity have been associated with a range of autoimmune diseases. However, the precise mechanism by which IL-18 induces such pathology remains unclear. In this study we provide direct evidence that IL-18 activates neutrophils via TNF-alpha induction, which drives the production of leukotriene B(4) (LTB(4)), which in turn leads to neutrophil accumulation and subsequent local inflammation. rIL-18 administered i.p. resulted in the local synthesis of LTB(4) and a rapid influx of neutrophils into the peritoneal cavity, which could be effectively blocked by the LTB(4) synthesis inhibitor MK-886 (MK) or its receptor antagonist CP-105,696. IL-18-induced neutrophils recruitment and LTB(4) production could also be blocked by a neutralizing anti-TNF-alpha Ab. In addition, IL-18 failed to induce neutrophil accumulation in vivo in TNFRp55(-/-) mice. In an IL-18-dependent murine collagen-induced arthritis model, administration of MK significantly inhibited disease severity and reduced articular inflammation and joint destruction. Furthermore, MK-886-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Finally, we showed that IL-18-activated human peripheral blood neutrophils produced significant amounts of LTB(4) that were effectively blocked by the MK. Together, these findings provide a novel mechanism whereby IL-18 can promote inflammatory diseases.     

Evidence for the involvement of eicosanoids in the regulation of normal body temperature

(1) In CD-1 mice and Sprague–Dawley rats, dark-induced elevation of body temperature (T_b) was accompanied by an increase in hypothalamic prostaglandin E₂ (PGE₂) production. (2) Indomethacin, a cyclooxygenase inhibitor, at a dose (5 mg/kg) that did not affect the daytime values of T_b, prevented the increase of T_b after the onset of darkness. (3) Pretreatment of mice with the inhibitor of leukotriene synthesis, MK-886 (1 mg/kg), prevented the decrease of daytime T_b. (4) The results suggest the involvement of PGE₂ and leukotrienes in the regulation of normal daily variations of T_b.     

Eicosanoids and their role in immune modulation in fish—a brief overview

Eicosanoids have been demonstrated to play a central role in immune regulation in mammals brought about by their direct effects on cells such as macrophages and lymphocytes or by their indirect effects via cytokines. Studies have shown that fish mononuclear phagocytes, granulocytes and thrombocytes synthesize and release both cyclooxygenase- and lipoxygenase-derived products such as prostaglandin E₂, leukotriene B₄ and lipoxin A₄. Whether lymphocytes have the ability to generate leukotrienes and lipoxins is still unclear but they do appear to have 12-lipoxygenase activity that leads to the generation of 12-hydroxy fatty acid derivatives. As in mammals, leukotriene and lipoxin biosynthesis requires the presence of a 5-lipoxygenase activating protein-like molecule that is sensitive to the action of the specific inhibitor, MK-886. The prostaglandin-generating ability of trout macrophages can be altered by incubation with lipopolysaccharide suggesting the possible presence of an inducible cyclooxygenase activity. Prostaglandins have been found to suppress the mitogen-induced proliferation of trout leucocytes and the generation of humoral antibody and plasma cells both in vivo and in vitro. The lipoxygenase products, leukotriene B₄ and lipoxin A₄ have more variable effects ranging from inhibition to stimulation depending on the assay system employed. Overall, there is clear evidence that eicosanoids play a role in immune regulation in fish in a similar way to that reported in mammals.     

COX-2-dependent and -independent biosynthesis of dihydroxy-arachidonic acids in activated human leukocytes

Biosynthesis of 5,15-dihydroxyeicosatetraenoic acid (5,15-diHETE) in leukocytes involves consecutive oxygenation of arachidonic acid by 5-lipoxygenase (LOX) and 15-LOX in either order. Here, we analyzed the contribution of cyclooxygenase (COX)-2 to the biosynthesis of 5,15-diHETE and 5,11-diHETE in isolated human leukocytes activated with lipopolysaccharide and calcium ionophore A23187. Transformation of arachidonic acid was initiated by 5-LOX providing 5S-HETE as a substrate for COX-2 forming 5S,15S-diHETE, 5S,15R-diHETE, and 5S,11R-diHETE as shown by LC/MS and chiral phase HPLC analyses. The levels of 5,15-diHETE were 0.45 ± 0.2 ng/10⁶ cells (mean ± SEM, n = 6), reaching about half the level of LTB₄ (1.3 ± 0.5 ng/10⁶ cells, n = 6). The COX-2 specific inhibitor NS-398 reduced the levels of 5,15-diHETE to below 0.02 ng/10⁶ cells in four of six samples. Similar reduction was achieved by MK-886, an inhibitor of 5-LOX activating protein but the above differences were not statistically significant. Aspirin treatment of the activated cells allowed formation of 5,15-diHETE (0.1 ± 0.05 ng/10⁶ cells, n = 6) but, as expected, abolished formation of 5,11-diHETE. The mixture of activated cells also produced 5S,12S-diHETE with the unusual 6E,8Z,10E double bond configuration, implicating biosynthesis by 5-LOX and 12-LOX activity rather than by hydrolysis of the leukotriene A₄-epoxide. Exogenous octadeuterated 5S-HETE and 15S-HETE were converted to 5,15-diHETE, implicating that multiple oxygenation pathways of arachidonic acid occur in activated leukocytes. The contribution of COX-2 to the biosynthesis of dihydroxylated derivatives of arachidonic acid provides evidence for functional coupling with 5-LOX in activated human leukocytes.     

Roles of superoxide and myeloperoxidase in ascorbate oxidation in stimulated neutrophils and H2O2-treated HL60 cells

Ascorbate is present at high concentrations in neutrophils and becomes oxidized when the cells are stimulated. We have investigated the mechanism of oxidation by studying cultured HL60 cells and isolated neutrophils. Addition of H₂O₂ to ascorbate-loaded HL60 cells resulted in substantial oxidation of intracellular ascorbate. Oxidation was myeloperoxidase-dependent, but not attributable to hypochlorous acid, and can be explained by myeloperoxidase (MPO) exhibiting direct ascorbate peroxidase activity. When neutrophils were stimulated with phorbol myristate acetate, about 40% of their intracellular ascorbate was oxidized over 20 min. Ascorbate loss required NADPH oxidase activity but in contrast to the HL60 cells did not involve myeloperoxidase. It did not occur when exogenous H₂O₂ was added, was not inhibited by myeloperoxidase inhibitors, and was the same for normal and myeloperoxidase-deficient cells. Neutrophil ascorbate loss was enhanced when endogenous superoxide dismutase was inhibited by cyanide or diethyldithiocarbamate and appears to be due to oxidation by superoxide. We propose that in HL60 cells, MPO-dependent ascorbate oxidation occurs because cellular ascorbate can access newly synthesized MPO before it becomes packaged in granules: a mechanism not possible in neutrophils. In neutrophils, we estimate that ascorbate is capable of competing with superoxide dismutase for a small fraction of the superoxide they generate and propose that the superoxide responsible is likely to come from previously identified sites of intracellular NADPH oxidase activity. We speculate that ascorbate might protect the neutrophil against intracellular effects of superoxide generated at these sites.     

Novel 1-(2-aminopyrazin-3-yl)methyl-2-thioureas as potent inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2)

Novel 1-(2-aminopyrazin-3-yl)methyl-2-thioureas are described as inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2). These compounds demonstrate potent in vitro activity against the enzyme with IC₅₀ values as low as 15 nM, and suppress expression of TNFα in THP-1 cells and in vivo in an acute inflammation model in mice. The synthesis, structure–activity relationship (SAR), and biological evaluation of these compounds are discussed.     

MK886, a potent and specific leukotriene biosynthesis inhibitor blocks and reverses the membrane association of 5-lipoxygenase in ionophore-challenged leukocytes

Recently, we have shown that ionophore activation of human leukocytes results in leukotriene synthesis and a translocation of 5-lipoxygenase from the cytosol to cellular membrane. This membrane translocation was postulated to be an important early activation step for the enzyme. 3-[1-(p-Chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2- dimethylpropanoic acid (MK886) is a potent and specific inhibitor of leukotriene biosynthesis in vivo and in intact cells, but has no direct effect on 5-lipoxygenase activity in cell-free systems. In this report, we show that MK886 can both prevent and reverse the membrane translocation of 5-lipoxygenase, in conjunction with the inhibition of leukotriene synthesis. Similar compounds of the indole class could also inhibit the membrane translocation of 5-lipoxygenase in a rank order of potency that correlated with their potencies for leukotriene synthesis inhibition. In contrast L-656,224, a direct 5-lipoxygenase inhibitor, had no effect on the translocation of the enzyme. Attempts to demonstrate the effects of MK886 on the association of 5-lipoxygenase with membrane in cell-free preparations failed due to a nonspecific Ca2+-dependent sedimentation of the enzyme. The mechanism of action of MK-886 is therefore to block translocation, prevent subsequent activation of 5-lipoxygenase, and hence block cellular leukotriene biosynthesis.     

A new method to evaluate anti-allergic effect of food component by measuring leukotriene B<Subscript>4</Subscript> from a mouse mast cell line

Leukotrienes (LTs), chemical mediators produced by mast cells, play an important role in allergic symptoms such as food allergies and hay fever. We tried to construct an evaluation method for the anti-LTB₄ activity of chemical substances using a mast cell line, PB-3c. PB-3c pre-cultured with or without arachidonic acid (AA) was stimulated by calcium ionophore (A23187) for 20 min, and LTB₄ production by the cells was determined by HPLC with UV detection. LTB₄ was not detected when PB-3c was pre-cultured without AA. On the other hand, LTB₄ production by PB-3c pre-cultured with AA was detectable by HPLC, and the optimal conditions of PB-3c for LTB₄ detection were to utilize the cells pre-cultured with 50 µM AA for 48 h. MK-886 (5-lipoxygenase inhibitor) completely inhibited LTB₄ production, but AACOCF₃ (phospholipase A₂ inhibitor) slightly increased LTB₄ production, suggesting that LTB₄ was generated from exogenous free AA through 5-lipoxygenase pathway. We applied this technique to the evaluation of the anti-LTB₄ activity of food components. PB-3c pre-cultured with 50 µM AA for 48 h was stimulated with A23187 in the presence of 50 µM soybean isoflavones (daidzin, genistin, daidzein, and genistein), equol, quercetin, or kaempferol. Genistein, equol, quercetin, and kaempferol strongly inhibited LTB₄ production without cytotoxicity. These results suggest that a new assay system using PB-3c is convenient to evaluate LTB₄ inhibition activity by food components. This method could be utilized for elucidation of the mechanisms of LTB₄ release suppression by food components such as flavonoids and the structure–activity relationship.     

Age-Dependent Relevance of Endogenous 5-Lipoxygenase Derivatives in Anxiety-Like Behavior in Mice

When 5-lipoxygenase (5-LO) is inhibited, roughly half of the CNS effect of the prototypic endocannabinoid anandamide (AEA) is lost. Therefore, we decided to investigate whether inhibiting this enzyme would influence physiological functions classically described as being under control of the endocannabinoid system. Although 5-LO inhibition by MK-886 reduced lipoxin A₄ levels in the brain, no effect was found in the elevated plus maze (EPM), even at the highest possible doses, via i.p. (10 mg/kg,) or i.c.v. (500 pmol/2 µl) routes. Accordingly, no alterations in anxiety-like behavior in the EPM test were observed in 5-LO KO mice. Interestingly, aged mice, which show reduced circulating lipoxin A₄ levels, were sensitive to MK-886, displaying an anxiogenic-like state in response to treatment. Moreover, exogenous lipoxin A₄ induced an anxiolytic-like profile in the EPM test. Our findings are in line with other reports showing no difference between FLAP KO or 5-LO KO and their control strains in adult mice, but increased anxiety-like behavior in aged mice. We also show for the first time that lipoxin A₄ affects mouse behavior. In conclusion, we propose an age-dependent relevancy of endogenous 5-LO derivatives in the modulation of anxiety-like behavior, in addition to a potential for exogenous lipoxin A₄ in producing an anxiolytic-like state.     

Involvement of eicosanoids in the hypothermic response to lipopolysaccharide during endotoxemia in rats

Hypothermia is one of the prominent features of the acute phase response to endotoxin (LPS). This study was undertaken to elucidate the effects of the COX-inhibitor Indomethacin (INDO) and the selective FLAP inhibitor MK-886 on LPS-induced hypothermia, mortality and increase in production of hypothalamic prostaglandin E(2) (PGE(2)) and leukotriene during endotoxemia.It has been demonstrated that INDO and MK-886 significantly attenuate the hypothermia induced by LPS, but MK-886 has a lesser (protective) effect than INDO. Only INDO was found to attenuate significantly the hyperthermic response to LPS. Furthermore, INDO significantly reduced the elevation in hypothalamic PGE(2) levels. MK-886 significantly reduced the elevation in hypothalamic leukotriene production only when LPS was given in a dose of 1mg/kg. Both drugs failed to reduce the elevation in plasma TNF-alpha and mortality induced by LPS.We conclude that in rats, febrile response to endotoxin involves many inflammatory mediators. However, it seems that PGE(2) and leukotrienes do not have a pivotal role in the mechanism of LPS-induced mortality.     

The priming effect of halogenated phospholipids on the functional responses of human neutrophils

Halogenated lipids formed in the reactions with myeloperoxidase (MPO)-derived species may contribute to the regulation of the functional activity of cells. In the present study we have investigated the effects of chloro- and bromohydrins formed in the HOCl and HOBr reactions, respectively, with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) on three different functional responses of human neutrophils: H₂O₂ generation, degranulation (MPO exocytosis), and cell aggregation. It was shown that POPC chloro- and bromohydrins (POPC-Cl and POPC-Br) induced the priming of neutrophils, resulting in significant upregulation of cell responses to neutrophil stimulators such as N-formyl-Met-Leu-Phe and lectin from Solanum tuberosum. The stimulating effects of POPC-Cl and POPC-Br were observed at low micromolar concentrations (liposomal concentration of POPC, 0.5–5 μM; the content of POPC-Cl or POPC-Br, 38 ± 3% of total lipids) after a short exposure (about 5 min) of the neutrophils to POPC-Cl or POPC-Br. These results suggest that halogenated lipids formed in vivo via MPO-dependent reactions may be considered as a new class of biologically active substances that are potentially able to contribute to the priming of myeloid cells in the sites of inflammation and serve as inflammatory response modulators.     

Development of smart cell-free and cell-based assay systems for investigation of leukotriene C4 synthase activity and evaluation of inhibitors

Cysteinyl leukotrienes (cys-LTs) cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A₄ (LTA₄) that is subsequently conjugated with glutathione (GSH) by LTC₄ synthase (LTC₄S). Cys-LT receptor antagonists and LTC₄S inhibitors have been developed, but only the former have reached the market. High structural homology to related enzymes and lack of convenient test systems due to instability of added LTA₄ have hampered the development of LTC₄S inhibitors. We present smart cell-free and cell-based assay systems based on in situ-generated LTA₄ that allow studying LTC₄S activity and investigating LTC₄S inhibitors. Co-incubations of microsomes from HEK293 cells expressing LTC₄S with isolated 5-LOX efficiently converted exogenous AA to LTC₄ (~ 1.3 μg/200 μg protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC₄S with Ca^2 +-ionophore A23187 and 20 μM AA resulted in strong LTC₄ formation (~ 250 ng/10⁶ cells). MK-886, a well-known 5-LOX activating protein (FLAP) inhibitor that also acts on LTC₄S, consistently inhibited LTC₄ formation in all assay types (IC₅₀ = 3.1–3.5 μM) and we successfully confirmed TK04a as potent LTC₄S inhibitor in these assay systems (IC₅₀ = 17 and 300 nM, respectively). We demonstrated transcellular LTC₄ biosynthesis between neutrophils or 5-LOX-expressing HEK293 cells that produce LTA₄ from AA and HEK293 cells expressing LTC₄S that transform LTA₄ to LTC₄. In conclusion, our assay approaches are advantageous as the substrate LTA₄ is generated in situ and are suitable for studying enzymatic functionality of LTC₄S including site-directed mutations and evaluation of LTC₄S inhibitors.     

Familial Mediterranian fever: clastogenic plasma factors correlated with increased O2 –– production by neutrophils

Familial Mediterranean fever (FMF) is an autosomal recessive disease predominantly affecting Armenians and non-Ashkenazi Jews. The disease begins in childhood with paroxysmal attacks of pain and fever accompanied by peritonitis, pleuritis, and synovitis. During the acute phase, there is a massive influx of polymorphonuclear leukocytes into the serosal membranes, connected with degranulation of the neutrophils and with secretion of lysosomal enzymes and pyrogenic substances. An increase in the lipoxygenase product, leukotriene B₄, a chemotactic agent, and a decrease in the activity of the inhibitor of chemotaxis, C5a, in serosal fluids have been considered responsible. Previous work from our laboratories had shown that the chromosomal instability observed in blood cultures of patients with FMF is secondary to circulating clastogenic factors (CFs), and that the antioxidant enzyme superoxide dismutase, as well as lipoxygenase inhibitors, reduce the chromosome damaging effects. CFs are observed in chronic inflammatory diseases and in various other pathological conditions accompanied by oxidative stress. Similar clastogenic materials were found in supernatants of neutrophils and monocytes after a respiratory burst and were shown to contain lipid peroxidation products and cytokines. In the present study we compared the clastogenic effects exerted by plasma ultrafiltrates from 20 adult patients with FMF to the unstimulated O₂ ^– production of their neutrophils. In comparison to 20 age- and sex-matched controls, which were studied simultaneously, the O₂ ^– production by patient’s neutrophils was routinely higher than that of controls. The clastogenic effects of patient’s plasma, expressed as the number of chromosomal aberrations induced in test cultures of healthy donors, were correlated with the importance of O₂ ^– production by their neutrophils (r = 0.5235). Even if the relative contribution of disturbance in arachidonic acid metabolism, neutrophil activation, and CF formation in the disease process remains unclear, the demonstration of oxidative stress in this genetic disorder suggests the use of antioxidants and free radical scavengers, in particular during acute attacks, when the classical colchicine treatment is without effect. Received: 15 June 1997 / Accepted: 18 July 1997     

Leukotriene synthesis inhibition and anti-ige challenge of human lung parenchyma

The leukotriene (LT) synthesis inhibitors BAY x1005 and MK-886 were evaluated in human lung parenchyma challenged with an anti-IgE. The anti-IgE-induced LTE₄ release was time- and dose-dependent. Treatment of the parenchyma with indomethacin (3 μM) prior to anti- IgE challenge inhibited the 6-keto prostaglandin F_1α (6-keto PGF_1α) release and enhanced (36%) the quantities of LTE₄ detected during IgE-stimulations. BAY x1005 and MK-886 were assessed in the presence of indomethacin (3 μM) and the IC₅₀ values for both inhibitors were similar (0.13 μM). BAY x1005 (1 μM) produced the same percent of inhibition of anti-IgE-induced LTE₄ release in the presence or absence of indomethacin. BAY x1005 (1 μM) did not alter the 6-keto PGF_1α release during anti-IgE challenge. The results indicate that BAY x1005 and MK-886 are potent inhibitors of LT synthesis when human lung parenchyma were stimulated by an anti-IgE.