TA-270 [4-Hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone], an Anti-Asthmatic Agent, Inhibits Leukotriene Production Induced by IgE Receptor Stimulation in RBL-2H3 Cells

Bronchial asthma is an atopic disease characterized by bronchoconstriction (O'Byrne et al., 1986), an influx of inflammatory cells into the airway (Kay, 1992), and bronchial hyper-responsiveness (Durham et al., 1988). One early event accompanied by these asthmatic responses is the activation of mast cells, which is elicited through the cross-linking of antigen-specific IgE bound to the high-affinity receptor for IgE (FcϵRI) (Kinet, 1989). Thus, antigen-induced stimulation of FcϵRI causes degranulation of the cells and release of inflammatory cytokines, including tumor necrosis factor-α (TNF-α) (Gordon and Galli, 1990) and phospholipase A₂ products such as prostaglandins and leukotrienes (Lewis et al., 1982; Holgate and Kay, 1985).

In the previous study (Aoki et al., 2000), we observed that a quinolinone derivative TA-270 [4-hydroxy-1-methyl-3-octyloxy-7-sinapinoylamino-2(1H)-quinolinone] inhibited antigen-induced asthmatic responses, including early-phase and late-phase bronchoconstriction, infiltration of inflammatory cells in bronchial lumen, and airway hyper-responsiveness in actively sensitized guinea pigs. The finding of the inhibition of early-phase bronchoconstriction by TA-270 raises the possibility that this drug might affect the mast cell functions because activation of this type of cell is important for the induction of early-phase airway responses (Metcalfe et al., 1997). Here we characterized the action mechanisms of TA-270 in RBL-2H3 mast cells. We found that this drug specifically inhibited production of leukotrienes, probably by inhibiting 5-lipoxygenase, a late-limiting enzyme for the synthesis of potent mediators for bronchoconstriction. The inhibitory action of TA-270 on leukotriene production was also observed in human neutrophils and eosinophils.

Materials and Methods

Drugs and Chemicals. TA-270 was synthesized by Dainippon Ink and Chemicals, Inc. Other chemicals were obtained from the following sources: RPMI 1640, fetal bovine serum, and O-dinitrophenol-conjugated human serum albumin (DNP-HSA) from Sigma-Aldrich (St. Louis, MO); DNP-specific monoclonal IgE from Zymed Laboratories, Inc. (San Francisco, CA); [¹⁴C]arachidonic acid and [³H]arachidonic acid from American Radiolabeled Chemicals, Inc. (St. Louis, MO); and p-nitrophenyl-β-d-hexosamine from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

Cell Cultures. RBL-2H3 mast cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum in a humidified air/CO₂ (19:1) atmosphere in 10-cm dishes. Two days before the experiments, cells were subcultured in 10-cm dishes for the measurement of ERK activity, in six multiplates for the experiments in which arachidonic acid metabolites were analyzed by TLC, and in 24 multiplates for a hexosaminidase release assay. The cells were subcultured in 12 multiplates for other experiments, including measurements of leukotriene release by immunoassay, TNF-α production, inositol phosphate production, and total release of arachidonic acid and its metabolites (without TLC analysis). In the experiments for inositol phosphate production, the medium was changed to an inositol-free RPMI 1640 medium containing 0.1% bovine serum albumin and 2 μCi/ml [³H]inositol 24 h before experiments as described previously (Kon et al., 1999). In the experiments for the release of arachidonic acid and its metabolites, [¹⁴C]arachidonic acid (0.3 μCi/well) was added into the medium for the experiments of TLC analysis and [³H]arachidonic acid (0.1 μCi/well) for the experiments without TLC analysis 10 h before experiments.

Assay Conditions of RBL-2H3 Cells with Test Agents. Except for the experiment of TNF-α production, which was performed in the same medium as the culture, i.e., RPMI 1640 supplemented with 10% fetal bovine serum, other experiments were performed in a Hepes-buffered medium consisting of 20 mM Hepes (pH 7.5), 134 mM NaCl, 4.7 mM KCl, 2 mM CaCl₂, 1.2 mM KH₂CO₃, 1.2 mM KH₂PO₄, 5 mM glucose, and 0.1% (w/v) bovine serum albumin (fraction V). Unless otherwise specified, the cells were treated with DNP-specific IgE (l μg/ml) 1 h before and with TA-270 at the appropriate concentration 30 min before stimulation with the antigen DNP-HSA and other stimulants.

Production of TNF-α, LTB_4, and Peptide Leukotrienes. The cells were incubated for the appropriate time as indicated, and the supernatant was collected. TNF-α was measured with the enzyme immunoassay kit system from Pierce Endogen (Rockford, IL), LTB_4, and peptide leukotrienes (including LTC₄, LTD_4, and LTE₄) were measured with the EIA system from Amersham (Buckinghamshire, UK) according to the manufacturer's instructions.

β-d-Hexosaminidase Release. After incubation with test agents for the indicated time, a supernatant was collected. The enzyme activity was measured as previously described (Hide et al., 1997). Briefly, an 80-μl test sample and 100 μl of 4 mM p-nitrophenyl-N-acetyl-β-glucosamine in 0.2 M citrate, pH 4.5, were incubated in 96-well plates to yield the chromophore, p-nitrophenol. The absorbance of the colored product was assessed at 405 nm using a microplate reader. The activity of β-hexosaminidase released into the medium was expressed as the percentage of total β-hexosaminidase, which was determined in the cells lysed in 0.2% Triton X-100.

Release of Arachidonic Acid and Its Metabolites from [³H] or [¹⁴C]Arachidonic Acid-Labeled Cells. For the measurement of the total release of [³H]arachidonic acid and its metabolites, the cells were incubated with test agents for 10 min. The supernatant was then collected, and its radioactivity was measured in a scintillation spectrometer. The activity was expressed as the percentage of the total radioactivity incorporated into the cells. In the case of the experiments for the analysis of the fate of [¹⁴C]arachidonic acid cleaved by phospholipase A₂, the supernatant was collected, and [¹⁴C]arachidonic acid and its metabolites were extracted and analyzed by TLC as described previously (Okajima and Ui, 1984). In brief, to the reaction supernatant (0.5 ml), 0.75 ml of chloroform, 0.75 ml of methanol, 0.2 ml of 2 M KCl, and 0.05 ml of 1 M HCl were added. The mixture was then shaken vigorously and separated into two phases by centrifugation. The lower phases were evaporated to dryness, and arachidonic acid and its metabolites were separated by TLC on a plate of silica gel 60 (Merck Ltd., Poole, Dorset, UK) with a solvent system of ethyl acetate/2,2,4-trimethylpentane/acetic acid/water (90:20:20:100, v/v, upper phase). The bands were visualized by autoradiography, and each lipid fraction was scraped and counted in a liquid scintillation spectrometer.

Measurement of Inositol Phosphate Production. The cells were incubated with test agents in the presence of 10 mM LiCl. The total inositol phosphates were separated as described previously (Kon et al., 1999).

Measurement of ERK Activity. RBL-2H3 cells were stimulated with the antigen for 5 min, and the reaction was then terminated by washing twice with ice-cold Ca²⁺- and Mg²⁺-free phosphate-buffered saline and adding 0.5 ml of a lysis buffer. The lysate was analyzed by Western blotting with an ERK-specific antibody to detect the change in gel mobility reflecting the phosphorylation of the enzyme as described previously (Sato et al., 1999).

Leukotriene Production in Human Neutrophils and Eosinophils. Human neutrophils and eosinophils were isolated from venous blood drawn from normal volunteers. The granulocyte fraction was prepared by sequential steps of dextran sedimentation, centrifugation through Ficoll-Paque, and hypotonic lysis of erythrocytes, as previously described (Borgeat et al., 1984). Granulocytes were used as neutrophils in this experiment. Eosinophils were isolated from the granulocyte fraction by negative selection with anti-CD16 microbeads and Super MACS, as previously described (Hansel et al., 1991). Human neutrophils and eosinophils were suspended in a 25 mM PIPES-buffered medium (pH 7.4) consisting of 110 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 0.6 mM MgCl₂, 5.4 mM glucose, and 5% fetal calf serum. Cells were treated with or without TA-270, stimulated with calcium ionophore A23187, and the reaction was stopped by the addition of the ice-cold PIPES-buffered medium, as previously described (Fukuda et al., 1995). The cell suspension was centrifuged, and the amount of LTB₄ and peptide leukotrienes in the supernatant was measured with a commercial EIA kit for neutrophils and eosinophils, respectively.

Cell-Free Assay for 5-Lipoxygenase. RBL-1 basophilic cell suspension (7 × 10⁷ cells/ml) in Ca²⁺- and Mg²⁺-free phosphate-buffered saline was sonicated and centrifuged at 100,000g at 4°C for 60 min. The supernatant was used for a cell-free 5-lipoxygenase activity. The crude enzyme extract was preincubated with test compounds for 4 min and incubated with [¹⁴C]arachidonic acid (0.5 μCi) in the presence of 1 mM CaCl₂ and 10 μM indomethacin for 30 min at 30°C at the final volume of 0.5 ml. The reaction was stopped by adding 1 ml of dichloromethane and 0.25 ml of 2 M formic acid. The mixture was then shaken and separated into two phases by centrifugation. The lower phase was evaporated to dryness and dissolved with ethanol. The ethanol solution was injected into a high performance liquid chromatograph column of Luna 3 μm C18(2) (Phenomenex Inc., Torrance, CA) eluted with acetonitrile/water/acetic acid (70: 30:1) at 1 ml/min and monitored by a radiomatic flow scintillation analyzer (PerkinElmer Life Sciences, Boston, MA). Arachidonic acid and a major 5-lipoxygenase product 5-HETE were detected at retention time approximately 11 and 3.9 min, respectively.

Data Presentation. All experiments were performed in duplicate or triplicate. The results of multiple observations were presented as means ± S.E.M. of at least three separate experiments unless otherwise stated. Statistical significance was assessed by Dunnett's multiple test.

Results

TA-270 Inhibits the Production of Leukotrienes but Hardly Affects Hexosaminidase Release and TNF-α Production. In Fig. 1, we measured the production of peptide leukotrienes, including leukotriene C₄, D_4, and E_4, in response to DNP-HSA in RBL-2H3 mast cells sensitized with the antigen-specific IgE. The antigen induced the time- (Fig. 1A) and dose-dependent (Fig. 1B) production of peptide leukotrienes. When the cells were treated with TA-270 at 10 μM, these responses were almost completely inhibited. The half-maximal concentration (IC₅₀) of TA-270 required for the inhibition of the response was about 3 μM (Fig. 1C).

In Fig. 2, we examined the effects of TA-270 on the degranulation response and TNF-α production. As an index of the degranulation response, we measured the activity of the hexosaminidase release. The enzyme was released in a manner dependent on time (Fig. 2A) and dose (Fig. 2B) in response to the antigen. Under the appropriate condition, the enzyme release reached 35 to 50% of the total enzyme. In contrast to the leukotriene production, however, TA-270 did not influence the degranulation response (Fig. 2, A and B). TA-270 also failed to significantly inhibit the antigen-induced TNF-α production (Fig. 2C).

Failure of TA-270 to Inhibit Early Signaling Events Involved in IgE Receptor-Mediated Actions. We next searched for the action site of TA-270 in the signaling pathways leading to the production of leukotrienes through stimulation of the IgE receptor. Since leukotrienes are phospholipase A₂ products, we examined the signaling events, which may regulate the enzyme activity. However, TA-270 was ineffective for inhibiting phospholipase C activation (Fig. 3A) and ERK activation (Fig. 3B) induced by IgE receptor stimulation. TA-270 was also ineffective for inhibiting phospholipase A₂ activity, which was estimated by the sum of the release of arachidonic acid and its metabolites (Fig. 3C).

Specific Inhibition by TA-270 of 5-Lipoxygenase Pathways. These results suggest that TA-270 may inhibit the metabolic pathway of arachidonic acid cleaved by phospholipase A₂ leading to leukotriene. In Fig. 4, we analyzed the fate of arachidonic acid. In the cells not stimulated with antigen, no significant band was detected. As examples, we presented the results of the effect of TA-270 on the basal accumulation of arachidonic acid or its metabolites in this figure (lanes 1 and 2). On the other hand, in the cells stimulated with antigen, several bands in addition to arachidonic acid were detected (lane 3). These bands, other than arachidonic acid, may be products of either 5-lipoxygenase or cyclooxygenase because the radioactivity of the respective band was decreased or disappeared by AA861 (an inhibitor of 5-lipoxygenase, Fig. 4D, lane 4), indomethacin (an inhibitor of cyclooxygenase, Fig. 4D, lane 8), or their combination (lane 9). The effects of the increasing concentration of TA-270 in the absence (lanes 5-7) and presence (lanes 10-12) of indomethacin were examined. The pattern of the distribution of arachidonic acid metabolites was very similar to that induced by AA861. For example, the radioactivity of some bands with an R_f value similar to that of 5-HETE or LTB₄ was significantly decreased by TA-270 (lane 7), and all the bands, except for arachidonic acid, almost completely disappeared in the presence of both indomethacin and TA-270 (lane 12). The results are summarized in Fig. 4B. Thus, TA-270 hardly affected the production of cyclooxygenase product PGD₂ but almost completely suppressed the formation of indomethacin-insensitive or 5-lipoxygenase products. At the expense of the inhibition of 5-lipoxygenase pathways, TA-270 treatment significantly increased the apparent formation of arachidonic acid.

In Fig. 4C, we examined whether TA-270 directly inhibits 5-lipoxygenase activity using a cell-free extract of RBL-1 basophilic cells, which express high 5-lipoxygenase activity (Kikuchi et al., 1994). To exclude the cyclooxygenase pathway, indomethacin was included in the assay medium. TA-270 and another 5-lipoxygenase inhibitor zileuton (Fig. 4D) inhibited the formation of the enzyme product 5-HETE in a dose-dependent manner. TA-270 was more potent than zileuton; the IC₅₀ value was 17 nM for TA-270 and 160 nM for zileuton.

Inhibitory Effects of TA-270 on Ca²⁺ Ionophore-Induced Leukotriene Release Not Only in RBL-2H3 Cells but Also Human Neutrophils and Eosinophils. The foregoing results suggest that the inhibitory action of TA-270 may no longer be restricted to the IgE receptor signaling. Actually, TA-270 inhibited the peptide leukotriene release as induced by Ca²⁺ ionophore A23187 with IC₅₀ of about 3 μM (Fig. 5C). Similar to the case of the IgE receptor stimulation, TA-270 never inhibited the Ca²⁺ ionophore-induced phospholipase A₂ activation (Fig. 5A) and hexosaminidase release (Fig. 5B).

The inhibition by TA-270 of A23187-induced production of leukotrienes was also observed in human neutrophils (LTB₄) and eosinophils (peptide leukotrienes), although the IC₅₀ values were approximately 0.3 μM (Fig. 5D) and 0.1 μM (Fig. 5E), respectively, which were about 10 times more potent than that in RBL-2H3 cells.

Discussion

In the present study, we showed that TA-270 inhibited IgE receptor-mediated accumulation of 5-lipoxygenase products without appreciable effects on phospholipase A₂ and cyclooxygenase activities in RBL-2H3 mast cells. TA-270 also hardly affected early signaling events involved in the IgE receptor-mediated actions. The inhibitory action of TA-270 on leukotriene production was also observed in human neutrophils and eosinophils. Thus, TA-270 inhibited the formation of not only 5-HETE but also leukotrienes, including LTB₄ and peptide leukotrienes in intact cells. These results suggest that this drug may inhibit 5-lipoxygenase activity. Indeed, TA-270 inhibited the enzyme activity in a cell-free extract of RBL-1 basophilic cells (Fig. 4C).

There was some difference in the potency of TA-270 to inhibit leukotriene production among cell types. It remains unknown whether this may reflect a difference in the ability of TA-270 to be incorporated into the cells or a difference in the drug sensitivity to the enzyme expressed in each cell. When 5-lipoxygenase is activated, the enzyme translocates from cytosol into the membrane and interacts with 5-lipoxygenase-activating protein (Dixon et al., 1990; Miller et al., 1990; Reid et al., 1990; Haeggstrom and Wetterholm, 2002). In our preliminary experiments, TA-270 did not appreciably affect the extent of the translocation of the enzyme into the membrane fraction in response to A23187. Thus, TA-270 appears to directly interact with 5-lipoxygenase and thereby inhibits the enzyme activity. Supporting this, TA-270 inhibited 5-lipoxygenase activity in a cell-free system.

In a previous in vivo study (Aoki et al., 2000), we showed that TA-270 inhibited antigen-induced asthmatic responses, including early-phase and late-phase bronchoconstriction, infiltration of inflammatory cells in bronchial lumen, and airway hyper-responsiveness in actively sensitized guinea pigs. An early airway response occurred within 1 min of the antigen stimulation in vivo, and the release of several mediators, including histamine and leukotrienes from mast cells, may be an important event (Metcalfe et al., 1997). TA-270 hardly affected the hexosaminidase release as an index of degranulation response from RBL-2H3 mast cells in the present study. Thus, inhibition of 5-lipoxygenase and the subsequent inhibition of leukotriene production may explain the inhibition of the early phase of bronchoconstriction in vivo.

In vivo, the inhibition patterns of TA-270 against the asthmatic responses were very similar to those of pranlukast, a potent peptide leukotriene receptor antagonist (Aoki et al., 2000). Thus, pranlukast inhibited not only the early-phase bronchoconstriction but also several late-phase responses, including late-phase bronchoconstriction, infiltration of inflammatory cells, and airway hyper-responsiveness (Nakagawa et al., 1993; Arakida et al., 2000). These results suggest that inhibition of leukotriene action may cause the inhibition of the late-phase responses as well as the early airway response. It is reasonable to speculate, therefore, that the inhibition of leukotriene production may also result in the inhibition of asthmatic responses regardless of the onset of the responses, in a manner similar to pranlukast. Actually, leukotrienes have been shown to be potent mediators for bronchoconstriction (Dahlen et al., 1980), plasma exudation (Peck et al., 1981), mucus secretion (Marom et al., 1982), and eosinophilic inflammation (Underwood et al., 1996), which may be involved in the early- and late-phase asthmatic responses (Coffey and Peters-Golden, 2003). Based on these pathological roles of leukotrienes in the asthmatic reactions, several drugs for inhibition of leukotriene action (receptor antagonists) and inhibition of leukotriene production (5-lipoxygenase inhibitor) are now available by prescription (Drazen et al., 1999; Krawiec and Wenzel, 2001; Coffey and Peters-Golden, 2003). However, at the present stage of investigation, it remains unknown whether the inhibition elicited by TA-270 of asthmatic responses in vivo can be attributed to the inhibition of 5-lipoxygenase.

In conclusion, TA-270 inhibited the production of leukotrienes induced by IgE receptor stimulation probably due to an inhibition of 5-lipoxygenase in inflammatory cells, including RBL-2H3 mast cells in vitro. The inhibition of leukotriene production may partially explain the mechanism of its anti-asthmatic actions in vivo.