Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The leukotrienes (LT) are proinflammatory mediators that play an important role in disease states such as asthma (Samuelsson, 1983; Lewis et al., 1990; Drazen et al., 1999) and have also been implicated as having a protective role in host defense against microbial infection (Demitsu et al., 1989; Bailie et al., 1996). LT synthesis from arachidonic acid (AA) is initiated by the Ca²⁺-dependent activation of 5-lipoxygenase (5-LO) (Rouzer and Samuelsson, 1985; Needleman et al., 1986), acting in concert with the AA-binding protein 5-LO-activating protein (Dixon et al., 1990), to form LTA₄. LTA₄ is the precursor for formation of the two major groups of LT, LTB₄ and the cysteinyl LT, LTC₄, D₄, and E₄.

Multiple mechanisms are recognized to regulate LT synthesis. These include the level of expression of LT-forming proteins (Bigby and Holtzman, 1987; Kargman and Rouzer, 1989; Coffey et al., 1994), their catalytic activity (Sporn and Peters-Golden, 1988), and their intracellular compartmentalization (Coffey et al., 1992; Brock et al., 1995; Cowburn et al., 1999). Catalytic activity of LT-forming proteins is known to be modulated by their phosphorylation by protein kinases. For example, activity of cytosolic phospholipase A₂ is increased by phosphorylation of serine 505 by protein kinase C or mitogen-activated protein kinase (MAPK) (Lin et al., 1993; Kramer et al., 1995; Borsch-Haubold et al., 1997). 5-LO activity is recognized to be primed by treatments that activate various protein kinases. Recently, phosphorylation of 5-LO by MAPK-activated protein kinase has been unequivocally demonstrated and shown to increase enzyme activity (Werz et al., 2000).

We have recently shown that 16-h pretreatment of alveolar macrophage (AM) with LPS reduced LT synthetic capacity (Coffey et al., 2000). This was explained, at least in part, by inducible nitric-oxide synthase (iNOS) and the generation of nitric oxide (NO) that directly inhibited 5-LO catalytic activity. In addition, the generation of reactive oxygen intermediates (ROI), which results from LPS treatment, also reduced cellular LT synthesis. The combination of NO and ROI results in the generation of peroxynitrite (ONOO) and caused a dose-dependent reduction in AM 5-LO metabolism (manuscript submitted for publication). Furthermore, treatment with ONOO resulted in nitrotyrosination of recombinant 5-LO and 5-LO in intact cells.

In the present study, we further examined the mechanisms by which ROI and ONOO caused a reduction in LT synthesis. Activation of the 5-LO enzyme is known to generate ROI, which in turn may inactivate the enzyme (Percival et al., 1992). Using the direct 5-LO inhibitor N-hydroxy urea A-64077 (zileuton), we attempted to block 5-LO activation and examined its effect on LT synthesis in LPS-treated cells. Zileuton blocked the suppression of 5-LO metabolism induced by overnight LPS in intact AM. We have demonstrated that one mechanism by which zileuton blocked LPS-induced suppression of LT synthesis was by inhibiting the nitrotyrosination of the 5-LO enzyme.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Cell Isolation and Culture. AM were obtained from 150-g specific pathogen-free female Wistar rats, as previously described (Peters-Golden et al., 1990). Lavaged cells were >98% AM after adherence, as determined by differential staining. Viability was >95%, as assessed by trypan blue exclusion. Isolated AM were resuspended in LPS-free DMEM (Invitrogen, Carlsbad, CA) at 0.5 × 10⁶/ml and plated as follows: 1 ml/well in 24-well plates for enzyme immunoassay (EIA), 1 ml/well in 24-well plates for high-performance liquid chromatography (HPLC) studies, and 5 ml/60-mm culture plate for immunoblot analysis. Cells were adhered for 1 h at 37°C in a humidified atmosphere of 5% CO₂, 95% air. Nonadherent cells were removed by washing twice with DMEM, and adherent cells were cultured in DMEM containing 10% fetal calf serum (Invitrogen), with or without various concentrations of agents for various time periods. Cell viability pre- and post-treatment was assessed by trypan blue exclusion.

Quantitation of 5-LO Metabolism in Intact Cells. Maximal capacity for 5-LO metabolism in intact cells was measured by EIA (Cayman Chemical) determination in cell-free supernatants of the predominant 5-LO product LTB₄. The cells were incubated with or without LPS (Escherichia coli serotype 0111:4B; Sigma Chemical, St. Louis, MO) for 16 h in the presence or absence of zileuton and/or L-NMMA. Cells were washed three times in DMEM and subsequently incubated with either Ca²⁺ ionophore A23187 (Calbiochem-Novabiochem) (1 µM) to stimulate release and metabolism of endogenous AA. The EIA results were confirmed by reverse phase HPLC analysis. Briefly, cells were prelabeled overnight with [³H]AA (PerkinElmer Life Science Products, Boston, MA) in the presence or absence of LPS. There was no effect of LPS on cellular uptake of radioactivity (data not shown). The eicosanoid profile was determined by HPLC analysis of ³H-radiolabeled eicosanoids [thromboxane B₂, prostaglandin E₂, LTB₄, and 5-hydroxyeicosatetraenoic acid (HETE)] released from A23187-stimulated cells, as described (Coffey et al., 1996). To assess total AA release, cells were stimulated in the presence of 0.1% bovine serum albumin (Sigma Chemical), which binds AA and prevents both metabolism and reacylation.

5-LO Cell-Free Assay. 5-LO activity of cell lysates (100 µg of total protein) or of purified recombinant 5-LO (gift from Denis Riendeau, Merck Frosst, Montreal, QC, Canada) was determined in 0.25 ml of 50 mM Tris containing 20 µM AA (Cayman Chemical) (including ~100,000 dpm of [³H]AA), 10 µM 13-(S)-hydroperoxy-9-cis-11-trans-octadecadienoic acid (Cayman Chemical) as activator, 0.6 mM CaCl₂, 0.1 mM EDTA, 0.1 mM ATP, and 12 µg/ml phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL), as described (Rouzer and Kargman, 1988; Coffey et al., 1998). After a 30-min incubation at room temperature the reaction was stopped by adding 1 ml of 30:4:1 (v/v/v) ether/MeOH/1 M citric acid, and samples were centrifuged at 1000g for 5 min. The upper phase was removed, evaporated under nitrogen, and stored at 70°C. Lipid residues were dissolved in 250 µl of acetonitrile and analyzed by HPLC on a 5-µm Bondapak C₁₈ column (30 × 0.4 cm; Waters, Milford, MA) by using a mobile phase of acetonitrile/water/trifluoroacetic acid, at a flow rate of 2 ml/min, as described (Coffey et al., 1992). Radioactivity in 1-ml eluate fractions was quantitated by on-line radioactivity detection. 5-LO specific activity was calculated based on conversion of AA to 5-hydroperoxyeicosatetraenoic acid/5-HETE plus LTB₄/LTB₄ isomers and was expressed as nanomoles per milligram of protein per 10 min.

Immunoblot Analysis of 5-LO and Nitrotyrosine. The relative quantities of cellular 5-LO, iNOS, and nitrotyrosine proteins were determined by Western blot analysis. Crude lysates were prepared, as described (Coffey et al., 1992), and subjected to SDS-polyacrylamide gel electrophoresis by the method of Laemmli (1970) on 10% acrylamide gels. Proteins were transferred overnight to nitrocellulose membranes and probed with rabbit polyclonal antibodies against human leukocyte 5-LO (1:3000 dilution) (provided by Dr. J. Evans, Merck Frosst) (Coffey et al., 1994) or iNOS (1:2000 dilution) (BIOMOL Research Laboratories, Plymouth Meeting, PA), or with anti-nitrotyrosine monoclonal antibody (1 µg/ml concentration) (Cayman Chemical). After washing, the blots were incubated for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Pharmacia Biotech, Arlington Heights, IL) at a dilution of 1:5000 for the polyclonal antibodies. For the monoclonal antibody the secondary used was a horseradish peroxidase-conjugated anti-murine IgG (Amersham Pharmacia Biotech) at a dilution of 1:5000. Membranes were then washed and incubated for 1 min with enhanced chemiluminescence detection reagents (Amersham Pharmacia Biotech) and exposed to film for varying time periods to ensure that densitometric quantitation was performed under conditions in which band density and exposure time were linearly related. Video densitometry was performed using NIH Image software (Scion, Frederick, MD).

Immunoprecipitation of 5-LO. Preswelled protein A-Sepharose (100 µl) (P-3391; Sigma Chemical) was transferred to 1.5-ml microcentrifuge tubes. Next, the beads were microcentrifuged at 16,000g for 5 s at 4°C, resuspended in 500 µl of ice-cold lysing buffer (10 mM Tris base, 300 mM NaCl, 1% Triton X-100, pH 7.2), microcentrifuged for 5 s, and again resuspended in 500 µl of lysing buffer. Antiserum (anti-human leukocyte 5-LO polyclonal antibody) (10 µl) was added to each tube and incubated with rotation for 3 h at 4°C. Nonimmune rabbit serum was used as a control. The AM were harvested, washed in ice-cold phosphate-buffered saline, counted, aliquoted into microcentrifuge tubes, microcentrifuged at 4°C for 5 s, resuspended in 500 µl of lysing buffer containing antiproteases (phenylmethylsulfonyl fluoride), vortexed for 3 s, and kept on ice for 30 min. The protein A-Sepharose and antibody were microcentrifuged for 5 s at 4°C, followed by the addition of 100 µl of lysing buffer containing antiproteases. Next, 500 µl of cells in lysing buffer was added, incubated for 2 h at 4°C, and microcentrifuged for 5 s at 4°C. The pellet was resuspended in 1 ml of lysing buffer and washed two times with lysing buffer and 10 mM Tris, pH 7.0, at 4°C. Sample buffer (2×, 45 µl) was added, boiled for 5 min, microcentrifuged for 5 s at 4°C, and the supernatant was transferred to 0.5-ml microcentrifuge tubes and frozen at 70°C.

Data Analysis. Where indicated, data were expressed as mean ± S.E.M. Differences between conditions were assessed by the paired Student's t test. Intergroup differences were analyzed by analysis of variance, with statistical significance assessed by Scheffé's test; p < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

LPS Activates 5-LO in Overnight Culture. We have recently shown that 16-h pretreatment of AM with LPS reduced the subsequent capacity for LT synthesis (Coffey et al., 2000). This was explained, at least in part, by the generation of NO and ROI, reactive species that alone and in combination suppress 5-LO metabolism (manuscript submitted for publication). We considered the possibility that in this experimental system LPS might activate 5-LO (Rankin and Harris, 1993). This might itself result in the generation of ROI that could inactivate the enzyme for subsequent stimulated LT synthesis. Indeed, 16-h incubation with LPS alone led to the accumulation of greater amounts of LTB₄ (4.6 ± 0.2 ng/ml) than did incubation with medium alone (3.6 ± 0.02 ng/ml), confirming activation of 5-LO.

Zileuton Abrogates LPS-Induced Suppression of 5-LO Metabolism. We sought to examine the effect of LPS pretreatment on AM 5-LO metabolism under circumstances where LPS activation of 5-LO was blocked. The reversible direct 5-LO inhibitor zileuton, added during the LPS pretreatment period, followed by zileuton removal by washing, prevented the LPS-induced suppression of 5-LO metabolism triggered by subsequent stimulation with A23187 (Fig. 1A). When the zileuton was not washed away prior to A21387 addition, it completely blocked stimulated LT synthesis (Fig. 1B). This ability of zileuton to abrogate LPS-induced suppression of LT synthesis was dose related, plateauing at 10 µM (Fig. 2). We have previously shown that LPS pretreatment had no effect on subsequent A23187-stimulated AA release (Coffey et al., 2000). Likewise, zileuton pretreatment had no effect on stimulated AA release (data not shown) or the profile of prostaglandin synthesis, as shown by HPLC analysis (Fig. 3). However, zileuton did result in an increase in 12-HETE. Interestingly, 16-h pretreatment with zileuton alone, followed by its removal, resulted in increased capacity for A23187-stimulated LT synthesis even in AM not incubated with LPS overnight, which is consistent with activation of the 5-LO enzyme after overnight incubation (data not shown). Furthermore, 16-h pretreatment of AM with zileuton, with and without LPS, increased 5-LO cell-free activity measured in crude cell lysates (Fig. 4). Similar protection from LPS-induced suppression was observed with the 5-LO inhibitor A63162 (Bell et al., 1992), but not with LTB₄ and cysteinyl-LT receptor antagonists (data not shown).

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Zileuton reverses the suppression of 5-LO metabolism in LPS-treated AM. A, AM were incubated for 16 h with or without LPS (1 µg/ml) in LPS-free DMEM containing 10% fetal calf serum. LPS-treated AM were treated with or without zileuton (10 µM) in parallel for 16 h, and the cells were washed and stimulated with A23817 (1 µM) for 30 min at 37°C. The medium was then analyzed for LTB4 by EIA. Data are expressed as picograms per milliliter of LTB4 and represent the mean ± S.E.M. from n = 5. ***p = 0.0001 between cells treated with and without LPS; p = 0.0001, n = 5 between LPS-treated cells and cells treated with LPS and zileuton. B, AM were incubated overnight, washed, and then stimulated with A23187 in the presence of zileuton. Data are expressed as LTB4 levels expressed as a percentage of untreated AM. ***p < 0.001, n = 3.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Zileuton dose dependently reverses LPS-induced suppression of AM LT synthesis. AM were incubated for 16 h with or without LPS (1 µg/ml) in LPS-free DMEM containing 10% fetal calf serum. LPS-treated AM were treated with or without increasing concentrations of zileuton (0.1-50 µM) in parallel for 16 h, and then the cells were washed and stimulated with A23817 (1 µM) for 30 min at 37°C and the medium was analyzed for LTB4 by EIA.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   HPLC analysis of zileuton's effect on AM eicosanoid profile. Prelabeled AM were incubated for 16 h with (B) or without (A) LPS (1 µg/ml). Prelabeled AM incubated with LPS were also treated with zileuton (C) in parallel for 16 h. The cells were washed and then were stimulated with A23187 (1 µM) for 30 min and [3H]AA metabolites identified by HPLC. Peaks were identified by coelution with authentic standards and the products expressed as a percentage of incorporated radioactivity. A representative profile of three separate experiments is shown.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Zileuton blocks the suppression of AM 5-LO cell-free activity after LPS treatment overnight. AM were incubated for 16 h with or without LPS (1 µg/ml) in LPS-free DMEM containing 10% fetal calf serum. AM treated with and without LPS were also treated with zileuton (10 µM) in parallel for 16 h, and the 5-LO cell-free assay performed on crude cellular lysates, as described under Materials and Methods. Data are expressed as a percentage of the value obtained for untreated AM cell lysate 5-LO cell-free activity (0.5 ± 0.04 nmol/mg/10 min). **p = 0.01 between cells treated with and without LPS, n = 3; p = 0.04 between LPS-treated cells and cells treated with LPS and zileuton, n = 3.

Interaction of Zileuton and L-NMMA. In our previous report we had determined that treatment with the L-arginine analog L-NMMA reversed the suppression of 5-LO metabolism elicited by overnight LPS treatment (Coffey et al., 2000), implicating NO in the process. We reasoned that if zileuton's effects were additive to those of L-NMMA, a mechanism of action independent of NO, such as prevention of ROI, generation would be suggested. However, zileuton treatment did not significantly add to the reversibility of L-NMMA to the suppression of 5-LO metabolism elicited by LPS (Fig. 5). This observation suggested that zileuton, too, may be acting at the level of iNOS or ONOO generation or effect.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of zileuton on L-NMMA-induced reversal of the suppression of 5-LO metabolism after LPS treatment. AM were incubated for 16 h with or without LPS (1 µg/ml) in LPS-free DMEM containing 10% fetal calf serum. LPS-treated AM were treated with or without zileuton (10 µM) or L-NMMA (100 µg/ml) in parallel for 16 h, and then the cells were washed and stimulated with A23817 (1 µM) for 30 min at 37°C and the medium was analyzed for LTB4 by EIA. Data presented are representative of three separate experiments.

Zileuton Blocks Nitrotyrosination of 5-LO. Zileuton had no effect on iNOS induction by LPS (data not shown). ONOO caused nitrotyrosination of 5-LO, as previously demonstrated (manuscript submitted for publication). Unexpectedly, incubation with zileuton blocked the nitrotyrosination of the purified recombinant 5-LO enzyme by ONOO (Fig. 6). The direct 5-LO inhibitor A63162 also blocked nitrotyrosination of 5-LO (data not shown), as did the nonspecific LO inhibitor and antioxidant nordihydroguaiaretic acid (NDGA) (Bell et al., 1992). The pretreatment of zileuton with increasing concentrations of ONOO at a ratio up to 400:1 (4 mM ONOO to 10 µM zileuton) did not block the ability of zileuton (10 µM) to subsequently block the nitrotyrosination of recombinant 5-LO by ONOO (500 µM). Pretreatment with zileuton also attenuated nitrotyrosination of 5-LO in intact AM treated with exogenous ONOO (Fig. 7). We next asked whether zileuton's ability to block nitrotyrosination was specific for 5-LO. Interestingly, zileuton also blocked ONOO-induced nitrotyrosination of BSA (Fig. 8), indicating that this represented a nonspecific scavenging effect independent of its 5-LO inhibitory capacity.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Zileuton blocks nitrotyrosination of recombinant 5-LO enzyme. Exogenous ONOO (500 µM) was added to recombinant 5-LO enzyme for 5 min in the presence or absence of zileuton (10 µM) and NDGA (50 nM and 10 µM). Western blot analysis detected nitrotyrosine at 78 kDa, which is the molecular mass for 5-LO. Data presented are representative of three separate experiments.

View larger version (42K): [in this window] [in a new window]   Fig. 7.   Zileuton blocks nitrotyrosination of 5-LO enzyme in intact AM. AM were incubated for 16 h with or without ONOO (500 µM) for the last hour in LPS-free DMEM containing 10% fetal calf serum in the presence or absence of zileuton (10 µM). 5-LO was immunoprecipitated from crude cellular lysates running equal cell numbers on a gel and probed for nitrotyrosine. ONOO treatment resulted in increased nitrotyrosination at 78 kDa, which corresponded to the molecular mass of 5-LO (top), and was confirmed when the blot was reprobed with anti-5-LO antibody (bottom). Nitrotyrosination of recombinant 5-LO is shown as a positive control. Data presented are representative of three separate experiments.

View larger version (47K): [in this window] [in a new window]   Fig. 8.   Zileuton blocks nitrotyrosination of BSA. Exogenous ONOO (500 µM) was added to BSA for 5 min, in the presence or absence of zileuton (10 µM), as described under Materials and Methods. Western blot analysis detected nitrotyrosine at 69 kDa, which is the molecular mass for BSA. Data presented are representative of three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

ONOO is a potent and relatively long-lived oxidant that may serve as an important cytotoxic agent in diseases such as acute lung injury (Haddad et al., 1994; Beckman et al., 1996). It would be expected to result from the NO and ROI generated in response to LPS, as previously demonstrated (manuscript submitted for publication). ONOO can cause nitrotyrosination of recombinant 5-LO enzyme and of 5-LO in intact cells (manuscript submitted for publication). This nitrotyrosination of the enzyme was associated with reduced 5-LO activity. We have identified a heretofore unidentified action of the 5-LO inhibitor zileuton. Our major findings are the following: 1) the direct 5-LO inhibitor zileuton dose dependently abrogated the suppression of 5-LO metabolism induced by overnight LPS treatment; 2) the effect of zileuton was not additive to L-NMMA in reversing LPS-induced suppression; 3) nitrotyrosination of 5-LO by ONOO is blocked by zileuton, but 5-LO nitrotyrosination, as detected by Western blot analysis, could not be demonstrated in macrophages after LPS treatment; and 4) the effect of zileuton on nitrotyrosination is independent of its 5-LO inhibitory capacity, indicating a direct scavenging effect.

ROI, such as superoxide, are formed in macrophages after stimulation by various biologically active substances, including LPS. These ROI may be generated not only through activation of NADPH oxidase but also by activation of the 5-LO enzyme. ROI have a biphasic concentration-dependent effect on 5-LO activity, with low doses being necessary for enzyme activity but higher doses causing inactivation (Denis et al., 1991; Percival et al., 1992). These effects reflect alterations in redox tone that influence the nonheme iron in the catalytic site of 5-LO. We observed that the reversible direct 5-LO inhibitor zileuton ameliorated the suppression of 5-LO metabolism in AM elicited by overnight LPS. Prevention of ROI generation during the LPS pretreatment period was considered to be a likely mechanism for zileuton's effects. However, the fact that the effect of zileuton was not additive to that of L-NMMA in reversing the suppressive effect of LPS on AM LT production suggested otherwise.

Zileuton had no effect on iNOS induction. We therefore considered the additional possibility that zileuton might abrogate ONOO-induced suppression of 5-LO enzymatic activity. Our previous investigations demonstrated that ONOO was capable of directly nitrotyrosinating the isolated 5-LO enzyme. Because 5-LO contains many tyrosine residues, including a number near the active site, this post-translational modification of 5-LO could explain the ability of ONOO to reduce cell-free enzyme activity. In this study we unexpectedly demonstrated that zileuton blocks direct nitrotyrosination of the recombinant 5-LO enzyme, as well as the 5-LO enzyme in intact cells. This unexpected action was not limited to zileuton, an N-hydroxyurea, because A63162, a related 5-LO inhibitor of the class hydroxamate (Bell et al., 1992), had a similar effect. The unrelated and nonspecific LO inhibitor NDGA was also effective. Because these agents are known to be reducing agents, quenching the oxidizing effect of ONOO could be one mechanism by which zileuton blocks nitrotyrosination by ONOO (Falgueyret et al., 1993). However, we were not able to overcome the reducing effect of zileuton with excess ONOO. Although zileuton does bind to the active site of the 5-LO enzyme, and by this mechanism could potentially block nitration of tyrosine residues, this is an unlikely explanation for its effect because it completely prevented nitrotyrosination of BSA by ONOO as well.

The possibility that zileuton's previously unrecognized capacity to scavenge ONOO could influence its effects on inflammatory processes will require further investigation. However, from what is known about ONOO and disease states it could be of significant clinical relevance. Disease states in which cytotoxic effects are associated with the expression of ONOO include atherosclerosis (Beckman and Koppenol, 1996), motor neuron disease (Estevez et al., 1998), and acute lung injury. Nitrotyrosination of lung tissue proteins occurs in animal models of sepsis and acute lung injury (Wizemann et al., 1994). Nitrotyrosination has also been demonstrated in the lungs of patients with acute lung disease (Haddad et al., 1994; Saleh et al., 1997; Tanaka et al., 1998), including adult respiratory distress syndrome (Sittipunt et al., 2001). In view of the association of nitrotyrosination of proteins with disease states, zileuton may play a protective role in preventing the cytotoxic effects of this agent.

In summary, we have demonstrated that the direct 5-LO inhibitor zileuton dose dependently abrogated the suppression of 5-LO metabolism induced by overnight LPS treatment through an inhibitory effect on the nitrotyrosination of the 5-LO enzyme. Because sepsis is associated with nitrotyrosination of the lung tissue (Sittipunt et al., 2001) and because nitrotyrosination of 5-LO is associated with reduced LT synthesis, preventing ONOO-induced nitrotyrosination may have utility in restoring LT synthetic capacity and thereby enhancing host defense capabilities in subjects surviving sepsis. As discussed in the Introduction, LT are important in host defense because they are present at sites of infection and they augment phagocytosis and killing of microorganisms (Demitsu et al., 1989; Bailie et al., 1996). The reduction in LT synthesis in AM after sepsis (Goya et al., 1992) may help to explain the observation that patients surviving sepsis are highly susceptible to the development of secondary pneumonia (Niederman and Fein, 1990; Zhang et al., 1998). Furthermore, it may be a useful tool in the treatment of a number of nonpulmonary diseases that involve nitrotyrosination of proteins in their pathogenesis.