Purification and characterization of leukotriene A4 epoxide hydrolase from dog lung

Leukotriene A₄ epoxide hydrolase from dog lung, a soluble enzyme catalyzing the hydrolysis of leukotriene A₄ (LTA₄) to leukotriene B₄ (LTB₄) was partially purified by anion exchange HPLC. The enzymatic reaction obeys Michaelis- Menten kinetics. The apparent Km ranged between 15 and 25 μM and the enzyme exhibited an optimum activity at pH 7.8. An improved assay for the epoxide hydrolase has been developed using bovine serum albumin and EDTA to increase the conversion of LTA₄ to LTB₄. This method was used to produce 700 mg of LTB₄ from LTA₄ methyl ester. The partial by purified enzyme was found to be uncompetitively inhibited by divalent cations. Ca²⁺, Mn⁺, Fe²⁺, Zn⁺² and Cu⁺² were found to have inhibitor constants (Ki) of 89 mM, 3.4 mM, 1.1 mM, 0.57 mM, and 28 μM respectively Eicosapentaenoic acid was shown to be a competitive inhibitor of this enzyme with a Ki of 200 μM. From these inhibition studies, it can be theorized that the epoxide hydrolae has at least one hydrophobic and one hydrophilic binding site.     

Effect of 6,9-deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin 1(1) (U-60,257), an inhibitor of leukotriene synthesis, on human neutrophil function

A23187, a calcium ionophore, stimulated a time-dependent generation of 5(S), 12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid (leukotriene B₄), production of superoxide anion (O₂^?) and release of granule-associated β-glucuronidase and lysozyme by human neutrophils. Leukotriene B₄ also elicited the selective release of granule enzymes from cytochalasin B-treated neutrophils. U-60,257, a recently identified inhibitor of leukotriene (LT) C₄ and D₄ synthesis, caused a dose-related (1–10 μM) suppression of LTB₄ production by A23187-activated neutrophils. Degranulation and O₂^? generation by neutrophils exposed to A23187 and the chemotactic oligopeptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP), were also inhibited with U-60,257.     

Inhibition of leukotriene B4-induced neutrophil degranulation by leukotriene B4-dimethylamide

Leukotriene B₄ (LTB₄) is a potent mediator of pro-inflammatory responses including neutrophil degranulation. Leukotriene B₄ dimethylamide has been synthesized and shown to inhibit neutrophil degranulation induced by LTB₄. The inhibition required time to develop (～60 secs), and had a K_D of circa 2 × 10^?7M, and occurred at concentrations where LTB₄ dimethylamide had negligible agonist activity.     

Biosynthesis, characterization and inhibition of leukotriene B4 in human whole blood

We have evaluated the biosynthesis, characterization and inhibition of Leukotrien (LT) B₄ in unstimulated and in A23187-stimulated human whole blood. LTB₄ was assayed by radioimmunoassay (RIA) both in unextracted serum and after extraction and thin-layer chromatography (TLC). Unstimulated human whole blood allowed to clot at 37°C for 60 min produced only trace amounts of LTB₄ (0.16±0.05 ng/ml, mean±SD, n=3). LTB₄-like immunoreactivity (ir-LTB₄) detectable in unstimulated serum samples was largely overestimated by direct RIA, most likely because of interfering substance(s) unrealed to cyclooxygenasep or lipoxygenase activity. Incubation of human whole blood with A23187 (2–10 μM) resulted in a concentration-dependent stimulation of LTB₄ production. At 10 μM A23187, ir-LTB₄ was 18±2.4 ng/ml (mean±SEM, n=28). In A23187-stimulated serum samples, LTB₄ concentrations measured by direct RIA correlated in a statistically significant fashion with those measured after extraction and TLC. Nafazatrom added caused a dose-dependent inhibition of A23187-stimulated ir-LTB₄ production with an IC₅₀ of 17 μM.     

Leukocytic functions in burn-injured patients

Anergy associated with an increase in suppressor helper T cell (T_c) ratio and a decrease in natural killer (NK) is one main cause of death following thermal injury (Tl). Recently, in vitro studies have shown that LTB₄ can induce human T_c to exert suppressor cell activity, and incubation of lymphocytes with LTB₄ for 24 hours significantly suppressed NK cell activity. Thus, we undertook an investigation of both AA metabolism and immunologic response in 20 patients who suffered 40–90% total body surface area (TBSA) burns. Cyclooxygenase (CO:RIA) and lipoxygenase (LO;HPLC det.) metabolites and superoxide (O₂^.−) production were measured in stimulated polymorphonuclear cells (PMNL) (A 23187 ± AA for icosanoid release; phorbol myristate acetate for O₂^.− production). Lyso-paf-acether (P-LPA) was measured in plasma samples. Ca²⁺-dependent K⁺ permeability in PMNL was measured by the cell K⁺ release induced by A 23187. T_c and T_c subsets were determined using monoclonal antibodies (OKT₃₊, OKT₄₊ and OKT₈₊). A biphasic sequential release of the different substances (leukocytic icosanoids and O₂^.− was monitored: increase ( 36–48 h after Tl) and decrease ( 72 h after Tl). The increase in AA stimulation was more transient than that of O₂^.−. The decline in the release of AA metabolites and O₂^.− production was associated with the anergic phase (decrease OKT₄₊/OKT₈₊ ratio) and with the clinical outcome of the patients. The decrease in LTB₄ and other LO metabolites could explain the impairment of neutrophil chemotaxis. Ca²⁺-dependent K⁺ permeability increased early up to 2 or 3 times normal.In order to go further with the mechanism of inhibition of LTB₄ and O₂^.− release, the effect of Tl plasma was assayed on normal leukocytes: a 10 min incubation with such plasma was sufficient to abolish LTB₄ secretion. A less important inhibition was observed with O₂^.− release (−32%) and Ca²⁺-dependent K⁺ permeability (−30%). Plasma inhibition seems to be due to a thermolabile factor(s) [protein(s): “suppressive factor(s) of membrane activation ”SFMA] which is (are) under active investigation using gel-filtration chromatography and fast protein liquid chromotography (FPLC). Among the SFMAs, certain acute phase proteins could play a key role: i.e., incubation (10 min) of normal PMNL with ceruloplasmin (1 mg/ml) abolished LO products and O₂^.− release.     

Leukotriene B4 binding to human neutrophils

[³H] Leukotriene B₄ (LTB₄) binds concentration dependency to intact human polymorophonuclear leukocytes (PMN's). The binding is saturable, reaches equilibrium in 10 min at 4°C, and is readily reversible. Mathematical modeling analysis reveals biphasic binding of [³H] LTB₄ indicating two discrete populations of binding sites. The high affinity binding sites have a dissociation constant of 0.46 × 10⁻⁹M and B_max of 1.96 × 10⁴ sites per neutrophil; the low affinity binding sites have a dissociation constant of 541 × 10⁻⁹M and a B_max of 45.6 × 10⁴ sites per neutrophil. Competitive binding experiments with structural analogues of LTB₄ demonstrate that the interaction between LTB₄ and the binding site is stereospecific, and correlates with the relative biological activity of the analogs. At 25°C[³H] LTB₄ is rapidly dissociated from the binding site and metabolized to 20-OH and 20-COOH-LTB₄. Purification of neutrophils in the presence of 5-lipoxygenase inhibitors significantly increases specific [³H] LTB₄ binding, suggesting that LTB₄ is biosynthesized during the purification procedure. These data suggest that stereospecific binding and metabolism of LTB₄ in neutrophils are tightly coupled processes.     

Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I₁ (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both $\text{in}$$\text{vitro}$ and $\text{in vivo}$. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB₄ synthesis in human peripheral neutrophils with an ID₅₀ of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.     

Production of leukotriene B4 and prostaglandin E2 by turbot (Scophthalmus maximus) leukocytes

Production of two eicosanoids derived from lipoxygenase and cyclooxygenase activities: leukotriene B₄ (LTB₄) and prostaglandin E₂ (PGE₂), respectively, have been simultaneously determined in turbot (Scophthalmus maximus) blood leucocyte and kidney macrophage supernatants by a reverse phase high performance liquid chromatography (HPLC) system coupled with a Diode–Array detector. Levels of LTB₄ after calcium ionophore challenge were 4.08 ng ml⁻¹ in blood leukocyte supernatants and 0.25 ng ml⁻¹ in kidney macrophage supernatants. The levels found for PGE₂ were 428.23 and 606.67 ng ml⁻¹ in blood leukocytes and kidney macrophage supernatants, respectively. When blood leukocytes were treated with the respective inhibitors for the enzymes implicated on the synthesis of both compounds an inhibition of 90.35% was observed for PGE₂ and 76.44% for LTB₄. The detection limit of the method was 0.15 ng ml⁻¹ for LTB₄ and 50 ng ml⁻¹ for PGE₂.     

Leukotriene B4 is a complete secretagogue in human neutrophils: a kinetic analysis

The interactions have been studied of leukotriene B₄ (LTB₄) and 20-COOH-LTB₄ with human neutrophils (PMN). Kinetic studies, utilizing continuous recording techniques, showed that LTB₄ activates PMN with respect to aggregation, mobilization of membrane-associated Ca²⁺, $\underset{\dot{}}{?}$ generation, and degranulation within seconds of exposure. Dose-response studies indicate 1) that LTB₄ is much more potent than its dicar?ylic acid derivative (20-COOH-LTB₄) or its all trans-isomer, and 2) that PMN responses to these agents are largely dependent upon pretreatment of the cells with cytochalasin B. These properties were similar to those of the microbial ionophores, ionomycin and A23187. Results demonstrate that LTB₄ rapidly activates PMN and indicate that LTB₄ serves as a complete secretagogue. Moreover, they provide additional evidence that oxidized fatty acids activate human PMN.     

Corticosteroid suppression of lipoxin A4 and leukotriene B4from alveolar macrophages in severe asthma

Background

An imbalance in the generation of pro-inflammatory leukotrienes, and counter-regulatory lipoxins is present in severe asthma. We measured leukotriene B₄ (LTB₄), and lipoxin A₄ (LXA₄) production by alveolar macrophages (AMs) and studied the impact of corticosteroids.

Methods

AMs obtained by fiberoptic bronchoscopy from 14 non-asthmatics, 12 non-severe and 11 severe asthmatics were stimulated with lipopolysaccharide (LPS,10 μg/ml) with or without dexamethasone (10^-6M). LTB₄ and LXA₄ were measured by enzyme immunoassay.

Results

LXA₄ biosynthesis was decreased from severe asthma AMs compared to non-severe (p < 0.05) and normal subjects (p < 0.001). LXA₄ induced by LPS was highest in normal subjects and lowest in severe asthmatics (p < 0.01). Basal levels of LTB₄ were decreased in severe asthmatics compared to normal subjects (p < 0.05), but not to non-severe asthma. LPS-induced LTB₄ was increased in severe asthma compared to non-severe asthma (p < 0.05). Dexamethasone inhibited LPS-induced LTB₄ and LXA₄, with lesser suppression of LTB₄ in severe asthma patients (p < 0.05). There was a significant correlation between LPS-induced LXA₄ and FEV₁ (% predicted) (r_s = 0.60; p < 0.01).

Conclusions

Decreased LXA₄ and increased LTB₄ generation plus impaired corticosteroid sensitivity of LPS-induced LTB₄ but not of LXA₄ support a role for AMs in establishing a pro-inflammatory balance in severe asthma.     

Bifunctional Lipocalin Ameliorates Murine Immune Complex-induced Acute Lung Injury

Molecules that simultaneously inhibit independent or co-dependent proinflammatory pathways may have advantages over conventional monotherapeutics. OmCI is a bifunctional protein derived from blood-feeding ticks that specifically prevents complement (C)-mediated C5 activation and also sequesters leukotriene B4 (LTB₄) within an internal binding pocket. Here, we examined the effect of LTB₄ binding on OmCI structure and function and investigated the relative importance of C-mediated C5 activation and LTB₄ in a mouse model of immune complex-induced acute lung injury (IC-ALI). We describe two crystal structures of bacterially expressed OmCI: one binding a C16 fatty acid and the other binding LTB₄ (C20). We show that the C5 and LTB₄ binding activities of the molecule are independent of each other and that OmCI is a potent inhibitor of experimental IC-ALI, equally dependent on both C5 inhibition and LTB₄ binding for full activity. The data highlight the importance of LTB₄ in IC-ALI and activation of C5 by the complement pathway C5 convertase rather than by non-C proteases. The findings suggest that dual inhibition of C5 and LTB₄ may be useful for treatment of human immune complex-dependent diseases.     

The Leukotriene B4/BLT1 Axis Is a Key Determinant in Susceptibility and Resistance to Histoplasmosis

The bioactive lipid mediator leukotriene B₄ (LTB₄) greatly enhances phagocyte antimicrobial functions against a myriad of pathogens. In murine histoplasmosis, inhibition of the LT-generating enzyme 5-lypoxigenase (5-LO) increases the susceptibility of the host to infection. In this study, we investigated whether murine resistance or susceptibility to Histoplasma capsulatum infection is associated with leukotriene production and an enhancement of in vivo and/or in vitro antimicrobial effector function. We show that susceptible C57BL/6 mice exhibit a higher fungal burden in the lung and spleen, increased mortality, lower expression levels of 5-LO and leukotriene B₄ receptor 1 (BLT₁) and decreased LTB₄ production compared to the resistant 129/Sv mice. Moreover, we demonstrate that endogenous and exogenous LTs are required for the optimal phagocytosis of H. capsulatum by macrophages from both murine strains, although C57BL/6 macrophages are more sensitive to the effects of LTB₄ than 129/Sv macrophages. Therefore, our results provide novel evidence that LTB₄ production and BLT₁ signaling are required for a histoplasmosis-resistant phenotype.     

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca²⁺ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB₄) demonstrated characteristic changes to cytoslic Ca²⁺ yielding an EC₅₀ of 4 nM. The pA₂ values for the specific LTB₄ receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca²⁺ in a dose dependant manner with pD₂ values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca²⁺ in response to LTB₄, FMLP and PAF with IC₅₀ values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca²⁺ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca²⁺ release in FMLP activated human neutrophils.     

A study of three vasodilating agents as selective inhibitors of thromboxane A2 biosynthesis

Three clinically efficacious vasodilatory drugs were found to be selective inhibitors of thromboxane A₂ biosynthesis. Hydralazine, dipyridamole, and diazoxide inhibited platelet aggregation at 1 × 10^?4 M, 1.75 × 10^?4 M, and 2 × 10^?3 M respectively. Their relative inhibitory potencies on thromboxane B₂ production in human platelet microsomes were examined and found to be similar to that observed for their inhibition on human platelet aggregation. At 10^?3 M, hydralazine, dipyridamole, and diazoxide inhibited thromboxane B₂ formation by 65 percent, 27 percent and 18 percent respectively. These compounds were examined in the sheep vesicular gland system, and they were shown not to be inhibitors of the cyclooxygenase enzyme. Thus, the inhibition of thromboxane A₂ biosynthesis by these three drugs in human platelet microsomes appeared to be specific at the thromboxane synthetase level.     

Evaluation of inhibitors of eicosanoid synthesis in leukocytes: Possible pitfall of using the calcium ionophore A23187 to stimulate 5′ lipoxygenase

The effect on arachidonate metabolism of two compounds (BW755C and benoxaprofen) which have been reported to inhibit 5′ lipoxygenase in leukocytes has been evaluated in human polymorphonuclear leukocytes (PMN) stimulated with the calcium ionophore A23187 and serum-treated zymosan (STZ). The syntheses of leukotriene B₄ (LTB₄) and thromboxane B₂ (TXB₂) from endogenous substrate were determined by specific radioimmunoassays as indicators of 5′ lipoxygenase and cyclo-oxygenase activity in the PMN respectively. Benoxaprofen inhibited the synthesis of leukotriene B₄ by human PMN stimulated with the calcium ionophore A23187, but it was approximately 5 times less potent than BW755C. However, benoxaprofen (IC₅₀ 1.6 × 10⁻⁴M) was approximately 100 times less potent than BW755C (IC₅₀ 1.7 × 10⁻⁶M) at inhibiting leukotriene B₄ synthesis induced by serum-treated zymosan. Both drugs inhibited thromboxane synthesis by leukocytes stimulated with A23187 or serum-treated zymosan at similar concentrations (approximately 5 × 10⁻⁶M). The data obtained using STZ as stimulus are consistent with previous studies and indicate that benoxaprofen is a relatively selective inhibitor of cylco-oxygenase. However, this selectivity was far less apparent when A23187 was used as a stimulus to release the eicosanoids which suggests that this inhibition could be via an indirect mechanism and therefore A23187 should be used with caution as a stimulus of 5′ lipoxygenase for evaluating inhibitors of eicosanoid synthesis.     

An Enzyme That Inactivates the Inflammatory Mediator Leukotriene B4 Restricts Mycobacterial Infection

While tuberculosis susceptibility has historically been ascribed to failed inflammation, it is now known that an excess of leukotriene A₄ hydrolase (LTA4H), which catalyzes the final step in leukotriene B₄ (LTB₄) synthesis, produces a hyperinflammatory state and tuberculosis susceptibility. Here we show that the LTB₄-inactivating enzyme leukotriene B₄ dehydrogenase/prostaglandin reductase 1 (LTB4DH/PTGR1) restricts inflammation and independently confers resistance to tuberculous infection. LTB4DH overexpression counters the susceptibility resulting from LTA4H excess while ltb4dh-deficient animals can be rescued pharmacologically by LTB₄ receptor antagonists. These data place LTB4DH as a key modulator of TB susceptibility and suggest new tuberculosis therapeutic strategies.     

Metabolism of leukotriene B4 in the monkey. Identification of the principal nonvolatile metabolite in the urine

Five milligrams of [5,6,8,9,11,12,14,15-³H₈]-leukotriene B₄ (LTB₄) (1.68 Ci/mmol) were infused into a monkey over a three hour period. Twenty-five per cent of the infused ³H-activity was recovered in the urine during the twenty hours of collection. Plasma and urinary metabolite volatility studies revealed that in contrast to previously studied eicosanoids, more than 70% per cent of the infused LTB₄³H-label was converted to tritiated water. The major nonvolatile urinary metabolite of LTB₄ representing 0.8% of the infused material was identified as 20-OH-LTB₄. LTB₄ was not excreted in the urine. Other nonvolatile metabolites of LTB₄ representing less than 0.4% each of the infused material were isolated from the urine. While there was an adequate quantity of some of these metabolites for partial characterization, there was insufficient material for structural elucidation. Further studies were performed in rabbits in which either LTB₄ or the structurally related compound 8,15-dihydroxyeicosatetraenoic acid (8,15-diHETE) were infused intravenously. In these rabbits the metabolism of LTB₄ and 8,15-diHETE was similar to that in the monkey with greater than 80% of the infused ³H-activity converted to tritiated water. These studies suggest that leukotriene B₄ and structurally related compounds undergo extensive degradation in vivo via the β-oxidation system.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opzonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B₄ and C₄. After 3–4 min, the levels of LTB₄ and LTC₄ were 93 and 35 pmol/310⁷ cells, respectively. These amounts were 2–4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC₄ were 8 and 20 times lower than those of LTB₄ after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

And THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB₄, LTC₄, and LTD₄. Incubation of these cells with recombinant human interferongamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB₄, LTC₄, and LTD₄ in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by thes cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (10000 units/ml)n and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37°C. The supernatants were deproteinated and assayed by RIA for LTB₄ and LTC₄ and by RP-HPLC for LTB₄, LTC₄, and LTD₄. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, <0.3ng/5 × 10⁶ cells. Peripheral blood mononuclear phagocytes from normal volumteers, cultured and challenged in vitro at under identical conditions, released 11.3 ± 2.9 ng LTB₄ and 2.0 ± 1.5 ng LTC₄/10⁶ viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n=3) nor by preincubation with PMA for 120 hours (n=3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.