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Aim: Glomerular infiltration of macrophages is a characteristic alteration of renal pathology in hyperlipidaemic renal injury. Leukotriene B4 (LTB4) is a bioactive eicosanoid and macrophage and has two key enzymes for LTB4 synthesis, 5-lipoxygenase and leukotriene A4 (LTA4) hydrolase. The purpose of this study was to evaluate the role of LTB4 in accelerated hyperlipidaemic renal injury., Methods: To induce accelerated hyperlipidaemic renal injury, 8 week old male spontaneously hypercholesterolaemic (SHC) rats were fed with a high cholesterol diet for 6 weeks. LTA4 hydrolase activities in the kidney and urine LTB4 levels were examined. The effects of LTB4 antagonist (ONO-4057) were also evaluated., Results: Urinary protein and LTB4 excretion was increased by a high cholesterol diet for 6 weeks. The scores of glomerular foam cell accumulation and sclerosis, numbers of infiltrated macrophages in glomeruli and interstitial area, LTA4 hydrolase activity in renal cortex were higher in the high cholesterol diet group than the normal diet group. LTB4 antagonist treatment reduced urinary protein and LTA4 activity and attenuated renal pathological changes., Conclusion: These results suggest that LTB4 directly contributes to accelerated hyperlipidaemic renal injury and the therapeutic potential of LTB4 antagonist for renal damage induced by hyperlipidaemia., (© 2011 The Authors. Nephrology © 2011 Asian Pacific Society of Nephrology.)

AS-35, (9-[4-acetyl-3-hydroxy-2-n-propylphenoxy) methyl]-3-(1H-tetrazol-5-yl)-4H-pyrido[1, 2-a] pyrimidin-4-one), was developed as a leukotriene (LT) receptor antagonist, which also inhibited IgE-mediated release of leukotrienes (LTs). We have investigated the action of AS-35 on the enzyme activities which are involved in the synthesis of LTC(4) and LTB(4) (LT-synthesizing enzymes); cytosolic phospholipase A(2) (cPLA(2)), 5-lipoxygenase (5-LO), leukotriene (LT)C(4) synthase and LTA(4) hydrolase. AS-35 dose-dependently inhibited IgE- and A23187-stimulated production of LTC(4) by up to 71.5-84.8% and that of LTB(4) by 48.3-49.2% at 2. 5x10(-5) M. The assays for cPLA(2)(-), 5-LO-, LTC(4) synthase- and LTA(4) hydrolase-activities revealed that the inhibition is attributable to suppression of cPLA(2), 5-LO and LTC(4) synthase but not LTA(4) hydrolase. We have also studied the action of AS-35 on the release of beta-hexosaminidase (beta-HEX) as a marker of preformed mediators. AS-35 had only weak inhibitory action on the release of beta-HEX. The results indicate that anti-allergic action of AS-35 is predominantly attributable to its inhibition of LT synthesis by suppressing three consecutive enzymes for LTC(4) synthesis.

We have observed an inhibitory action of magnolol on the production of leukotriene (LT) C4 and LTB4, important lipid mediators in allergy and inflammation. IgE- and A23187-stimulated production of LTC4 and LTB4 was measured by radio-immunoassay (RIA) in the absence or presence of various concentrations of magnolol in intact rat basophilic leukemia (RBL)-2H3 cells. Magnolol dose-dependently inhibited synthesis of LTC4 and LTB4. Magnolol inhibited the IgE-mediated increase of intracellular calcium ion concentration, resulting in the inhibition of cytosolic phospholipase A2 (cPLA2) and possibly 5-lipoxygenase (5-LO), both calcium ion-dependent enzymes. In cell-free studies magnolol inhibited LTC4 synthase activity. LTA4 hydrolase activity was only inhibited at the higher concentration (2.5 x 10(-5)M). These results indicate that magnolol inhibits production of LTs by inhibiting PLA2, 5-LO, LTC4 synthase and LTA4 hydrolase which are essential for LT-synthesis. Magnolol may have anti-allergic effect by blocking LT-synthesis.

There are substantial numbers of reports showing that leukotrienes (LTs) play important roles in adult asthma. No definite evidence has been demonstrated that LTs are involved in asthma attacks in children, although it is highly expected. In this report, we demonstrated that the levels of LTB4 and LTC4 but not thromboxane B2 (TXB2), a stable metabolite of TXA2, were significantly elevated in the bronchoalveolar lavage fluid, which was obtained from intubated and mechanically ventilated children with severe asthma attacks. This is direct evidence that LTB4 and LTC4 predominantly participate in asthma attacks in pediatric patients.

We examined the action of Shinpi-To (Formula divinita; TJ-85), a granular extract of seven Chinese medicinal herbs that is used in treating childhood asthma, on the leukotriene synthesis in rat basophilic leukemia-2H3 cells (RBL-2H3 cells). IgE-loaded cells were stimulated with anti-IgE serum in the presence or absence of Shinpi-To. Released LTC4 and LTB4 were measured by radioimmunoassay (RIA). Shinpi-To significantly inhibited IgE-mediated synthesis of leukotriene (LT)C4 and LTB4. To identify the inhibitory sites, we investigated the action of this extract on four synthetic enzymes, phospholipase A2 (PLA2), 5-lipoxygenase (5-LO). LTC4 synthase, and LTA4 hydrolase. Shinpi-To inhibited the A23187-stimulated release of [3H]arachidonic acid (AA) from the cell membrane, reflecting an effect on PLA2 activity. It also suppressed production of LTC4 and LTB4 when cell lysates were incubated with AA as substrate. It did not inhibit the production of LTC4 and LTB4 when LTA4-free acid was used as the substrate. Shinpi-To did not inhibit the IgE-mediated increase of intracellular Ca2+ ([Ca2+]i) concentration. Results indicate that Shinpi-To inhibits LT synthesis by inhibiting PLA2 and 5-LO activities without affecting the mobilization of [Ca2+]i.

We investigated the effects of Saiboku-to, Syoseiryu-to and compornent herbs of these two Kampo-Medicines (Saiko, Hange, Bukuryo, Ogon, Koboku, Taiso, Ninjin, Kanzo, Soyo, Syokyo, Keihi, Gomisi, Saisin, Syakuyaku, Mao and Kankyo) on the production of peptide leukotrienes (LTs) and LTB4 in cultured rat basophilic leukemia (RBL)-1 cells. Cultured RBL-1 cells were stimulated with Ca ionophore, A23187, at 10(-5) M in the absence or presence of various concentrations of these substances, and the production of peptide LTs and LTB4 was measured by reversed phase-high performance liquid chromatography (RP-HPLC). The production of LTs was dose-dependently suppressed by the addition of Saibokuto. Saiboku-to (100 micrograms/ml) showed 35% and 30% inhibition on the production of peptide LTs and LTB4, respectively. These inhibitory actions of Saiboku-to on LT-synthesis were attributable to the effects of its component herbs, Ogon, Koboku and Kanzo. On the other hand, Syoseiryu-to showed no inhibitory action on LT-production. these results indicate that anti-allergic action of Saiboku-to is, at least in part, attributable to its inhibitory action on LT-synthesis.

Background: Azelastine, oxatomide, and ketotifen are used for patients with allergic diseases. These drugs inhibit the release of chemical mediators including the leukotrienes; however, the mechanism involved is unclear., Objective: To clarify the mechanism of inhibition, we investigated the effects of three drugs on the function of phospholipase A2, 5-lipoxygenase, leukotriene C4 synthase, and leukotriene A4 hydrolase, which are all catabolic enzymes involved in synthesizing leukotriene C4 and leukotriene B4 in rat basophilic leukemia (RBL)-1 cells., Methods and Results: The production of leukotriene C4 and leukotriene B4 was measured by high performance liquid chromatography (HPLC). All three drugs inhibited the production of leukotriene C4 and leukotriene B4 when cells were stimulated with A23187. All three drugs also inhibited the A23187-stimulated release of 3H-arachidonic acid from membrane phospholipids. Azelastine inhibited the production of leukotriene C4, but not leukotriene B4, when either arachidonic acid or leukotriene A4 free acid was used as the substrate in our cell free system. Oxatomide and ketotifen did not inhibit the synthesis of either leukotriene C4 or leukotriene B4 in the same cell free study., Conclusion: Results indicated that oxatomide and ketotifen inhibit the production of leukotriene C4 and leukotriene B4 by inhibiting phospholipase A2 activity, whereas, azelastine inhibits the leukotriene C4 production by inhibiting phospholipase A2 and leukotriene C4 synthase.

Concentrations of immunoreactive leukotriene C4 (LTC4) and leukotriene B4 (LTB4) in the cerebrospinal fluid from 18 patients with aseptic meningitis, including 2 patients with encephalitis and 4 patients with febrile seizures, were measured by a sensitive and specific radioimmunoassay; these results were compared with those from control subjects. The concentrations of both LTC4 and LTB4 were elevated significantly in patients with meningitis (LTC4: 115.6 +/- 47.7 pg/ml; LTB4: 1,603.0 +/- 273.5 pg/ml; n = 18) compared to controls (LTC4: 83.2 +/- 21.6 pg/ml; LTB4: 1,219.3 +/- 161.5 pg/ml; n = 12; P < .05 and P < .01, respectively). However, there was no significant increase in LT levels in patients with febrile seizures. These findings suggest that LTs may play an important role in the inflammatory response induced by viral infections of the central nervous system.

To determine the regulatory mechanism of Leukotriene (LT) B4 synthesis by cytokines, we investigated the regulation of LTB4 generation by short-term (30 min) priming and long-term (15 h) enzyme-inducing actions of the four cytokines interleukin (IL)-3, IL-5, tumor necrosis factor alpha (TNF-alpha), and transforming growth factor alpha (TGF-alpha) in rat basophilic leukemia-1 (RBL-1) cells. Pretreatment of cells with IL-3 or IL-5 for 30 min increased A23187- (5x10(-9)M) stimulated synthesis of LTB4 by three to four times over control levels. However, IL-3 or IL-5 lacked this effect when stimulated with exogenous arachidonic acid A at 10(-4)M. TNF-alpha and TGF-alpha had no priming effect on LTB4 synthesis following stimulation with either A23187 (5x10(-9)M) or AA(10(-4)M). Stimulation with the calcium ionophore (A23187)(10(-5)M) or AA(10(-4)M) following 15-h exposure to these cytokines had no effect. These results suggest that IL-3 and IL-5 increase the production of LTB4 by priming the activity of phospholipase A2(PLA2) without inducing enzymes in the arachidonate 5-lipoxygenase pathway. Such a priming effect may be important in regulating the development of allergic and other diseases involving the inflammatory reaction.

The role of LTB4 in Kawasaki disease as a chemo-attractant and immunomodulator is reviewed through our own experience and reports by other investigators. In our experiment using 19 patients, we measured calcium ionophore-stimulated LTB4 synthesis in PMNs obtained in three different stages of the illness (acute, convalescent and recovered phases). LTB4 synthesis was significantly increased in the convalescent phase of the illness. Other investigators showed increased serum-LTB4 concentration in acute as well as convalescent phases, suggesting that LTB4 participated in the inflammatory process of Kawasaki disease as an inflammatory mediator and immunomodulator. However, no difference was found in LTB4 synthetic activity in PMNs in any phases of the illness between the patients with and without coronary lesions, which indicated that LTB4 was not a parameter of coronary aneurysm formation. Therapeutic use of high-dose gamma-globulin showed a tendency to decreased LTB4 synthesis in PMNs, although it is not conclusive.

It is postulated that inflammatory cells play an important role in the process of developing vasculitis in Kawasaki Disease. LTB4, a 5-lipoxygenase product of arachidonic acid is one of the most potent chemoattractants to inflammatory cells and is produced in large amounts by PMNs. We investigated the role of PMN-derived LTB4 in Kawasaki Disease. Isolated PMNs were obtained from 19 Kawasaki disease patients in three different phases of the illness, (acute phase: 0-12th day, convalescent phase: 13-29th day, restored phase: greater than 30th day). LTB4 synthesis in the convalescent phase was 26.43 +/- 4.2 ng/5 x 10(6) cells, which was significantly higher than those in the acute and restored phases (11.90 +/- 1.91, 13.87 +/- 1.86 ng) and also higher than that in the control subjects (10.65 +/- 1.26 ng: p less than 0.05). No differences were found between two groups with or without coronary lesions. The results imply that activated PMNs during the convalescent phase of illness produce larger amount of LTB4 which may participate in the development of inflammatory process of the disease and that PMN-derived LTB4 plays no significant roles in the development of coronary lesions.

Chronic neutrophilic leukemia (CNL) is a rare type of leukemia. We diagnosed a 81-year-old woman as CNL because she showed that sustained leukocytosis dominated by mature neutrophils, hepatosplenomegaly, high neutrophilic alkaline phosphatase (NAP) score, absence of the Ph1 chromosome and no evidence of leukemoid reaction. During the clinical course, she did not manifest hemorrhagic tendency or infection. We also examined neutrophilic function including chemotaxis, chemiluminescence, nitroblue tetrazolium (NBT) dye reduction, which all indicated normal neutrophil function. Using a reversed phase-high pressure liquid chromatography (HPLC), we detected the production of leukotriene B4 (LTB4) in neutrophils. We found that the LTB4 production was decreased in neutrophils whereas they showed normal chemotaxis. This discrepancy has never, to our knowledge, been reported before in case of CNL.