5-Lipoxygenase Activity, Respectively Calcium Influx and Suppressing

The Journal of Immunology

Natural Forms of Vitamin E and 139-Carboxychromanol, a Long-Chain Vitamin E Metabolite, Inhibit Leukotriene Generation from Stimulated Neutrophils by Blocking Calcium Inﬂux and Suppressing 5-Lipoxygenase Activity, Respectively

Ziying Jiang, Xinmin Yin, and Qing Jiang

Leukotrienes generated by 5-lipoxygenase (5-LOX)–catalyzed reaction are key regulators of inﬂammation. In ionophore- stimulated (A23187; 1–2.5 mM) human blood neutrophils or differentiated HL-60 cells, vitamin E forms differentially inhibited

leukotriene B4 (LTB4) with an IC50 of 5–20 mMforg-tocopherol, d-tocopherol (dT), and g-tocotrienol, but a much higher IC50 for a-tocopherol. 139-Carboxychromanol, a long-chain metabolite of dT, suppressed neutrophil- and HL-60 cell-generated LTB4 with an IC50 of 4–7 mM and potently inhibited human recombinant 5-LOX activity with an IC50 of 0.5–1 mM. In contrast, vitamin E forms had no effect on human 5-LOX activity but impaired ionophore-induced intracellular calcium increase and calcium inﬂux as well as the subsequent signaling including ERK1/2 phosphorylation and 5-LOX translocation from cytosol to the nucleus, a key 2+ event for 5-LOX activation. Further investigation showed that dT suppressed cytosolic Ca increase and/or LTB4 formation triggered by ionophores, sphingosine 1-phosphate, and lysophosphatidic acid but not by fMLP or thapsigargin, whereas 139-

carboxychromanol decreased cellular production of LTB4 regardless of different stimuli, consistent with its strong inhibition of the 5-LOX activity. These observations suggest that dT does not likely affect fMLP receptor-mediated signaling or store depletion- induced calcium entry. Instead, we found that dT prevented ionophore-caused cytoplasmic membrane disruption, which may account for its blocking of calcium inﬂux. These activities by vitamin E forms and long-chain carboxychromanol provide potential molecular bases for the differential anti-inﬂammatory effects of vitamin E forms in vivo. The Journal of Immunology, 2011, 186: 1173–1179.

eukotrienes are generated by activated leukocytes via 5- depletion of endoplasmic reticulum (ER) Ca2+ storage (6), or

by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. lipoxygenase (5-LOX)–catalyzed oxidation of arachidon- fMLP, which triggers receptor-mediated calcium release from ER L ic acid (AA). Leukotriene B4 (LTB4) and leukotriene C4 storage via a phospholipase C-mediated mechanism (7). In addi- (LTC4), produced by neutrophils and eosinophils, respectively, are tion, it has been shown that sphingosine-1 phosphate (S1P) and important lipid regulators of inflammation (1, 2). When leukocytes lysophosphatidic acid (LPA), two important lipid mediators, ac- are stimulated, 5-LOX, which is primarily located in the cytosol tivate intracellular calcium increase in neutrophils (8, 9). Given under resting condition, translocates to the membrane of the nu- the regulatory role of leukotrienes, modulation of their produc- cleus where it interacts with 5-LOX activating protein to form the tion via potentiating 5-LOX activity or Ca2+-related signaling functionally active enzyme (3). The translocation of 5-LOX is may have profound effects on inflammation and inflammation-as- triggered by intracellular calcium increase, which subsequently sociated diseases. http://classic.jimmunol.org activates downstream signaling including protein kinase C (PKC) Previous studies suggest that some vitamin E isoforms may and ERK (4, 5). Ca2+ release can be stimulated by calcium ion- be capable of modulating leukotriene formation. Natural forms ophores such as A23187, thapsigargin (THAP), which induces of vitamin E comprise eight lipophilic antioxidants; that is, a-, b-, g-, and d-tocopherol (aT, bT, gT, and dT) and a-, b-, g-, and d-tocotrienol (aTE, bTE, gTE, and dTE) (Fig. 1). In Department of Foods and Nutrition, Interdepartmental Nutrition Program, Purdue A23187-stimulated neutrophils, aT, the predominant form of University, West Lafayette, IN 47907

Downloaded from vitamin E in tissues, has been reported to enhance 5-LOX– Received for publication July 13, 2010. Accepted for publication November 16, catalyzed LTB at low micromolar concentrations but to sup- 2010. 4 press LTB at higher concentrations (10). aT is shown to de- This work was supported in part by National Institutes of Health Grants 4 R01AT001821, P01AT002620, and R21CA133651. crease LPS-stimulated LTB4 from human monocytes (11). Sup- Address correspondence and reprint requests to Qing Jiang, Department of Foods and plementation of aT appears to reverse aT deficiency-caused Nutrition, Purdue University, Stone Hall G1A, 700 W. State Street, West Lafayette, enhancement of LTB4 production in neutrophils (12, 13) but IN 47907. E-mail address: [email protected] inconsistently modulates LTB4 release under aT-sufficient con- Abbreviations used in this article: AA, arachidonic acid; 139-COOH, 139-carboxy- ditions (12, 14). Besides aT, we have demonstrated that gT, chromanol; COX, cyclooxygenase; FOX, ferrous oxidation–xylenol orange; 5-LOX, the major form of vitamin E in the United States diet, suppres- 5-lipoxygenase; LPA, lysophosphatidic acid; LTB4, leukotriene B4;LTC4, leukotriene C4; PI, propidium iodide; PKC, protein kinase C; S1P, sphingosine 1-phosphate; sed inflammation-enhanced leukotriene generation in several aT, bT, gT, or dT, a-, b-, g-, or d-tocopherol; aTE, bTE, gTE, or dTE, a-, b-, g-, or inflammation models in rodents (14–16). Despite these obser- d-tocotrienol; THAP, thapsigargin. vations, the mechanisms underlying these modulatory effects Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 are not completely understood. It is also not clear whether

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002342 1174 VITAMIN E AND ITS LONG-CHAIN METABOLITE INHIBIT LEUKOTRIENES

different forms of vitamin E show differential effects on 5-LOX– Assessment of 5-LOX activity mediated reactions. In this study, we investigated the effect Potential effects on the activity of 5-LOX were evaluated using the ferrous and mechanism of different forms of vitamin E on leukotriene oxidation–xylenol orange assay (FOX assay) as previously described (24). formation from human neutrophils or neutrophil-like differenti- Briefly, human recombinant 5-LOX (9 U) was preincubated with vitamin E ated HL-60 cells and eosinophil-like differentiated clone 15 HL- forms, 139-COOH, or zileuton (a specific 5-LOX inhibitor) for 4 min at 60 cells. In addition, in light of our recent studies that 139- room temperature in 50 mM Tris-HCl buffer (pH 7.4) containing 0.4 mM CaCl2. Reactions were initiated by the addition of AA (final 75 mM). Four carboxychromanol (139-COOH) (Fig. 1), a long-chain metabolite minutes later, the reaction was terminated by addition of FOX reagent of dT, competitively inhibited cyclooxygenases (cyclooxygen- containing 25 mM sulfuric acid, 100 mM xylenol orange, and 100 mM ase [COX]-1 and COX-2) (17), we also examined whether this ferrous sulfate dissolved in methanol/water (9:1). After color development, vitamin E metabolite has any effect on 5-LOX–catalyzed re- the absorbance was measured at 560 and 575 nm using a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA). actions. Intracellular calcium measurement Materials and Methods Differentiated HL-60 cells were incubated with 2 mM fluo-4 AM in Ca2+- Chemicals and Abs free HBSS in the dark at room temperature for 30 min (25). Cells were then washed twice and resuspended in HBSS buffer. In some studies, 3 3 Vitamin E forms, A23187, ionomycin, S1P, fMLP, DMSO, and protease 106 cells were incubated with vitamin E at 37˚C for 10 min in HBSS with inhibitor mixture were purchased from Sigma (St. Louis, MO). LPA was Ca2+ and then stimulated. To study Ca2+ influx, cells were incubated with obtained from Enzo Life Sciences (Farmingdale, NY). Zileuton was pur- vitamin E for 10 min in Ca2+-free HBSS, followed by stimulation with chased from Tocris Cookson (St. Louis, MO). Fluo-4 AM, RPMI 1640 A23187 or S1P for 1.3 min and Ca2+ added (final 1.25 mM). Intracellular medium, DMEM, and HBSS buffer were purchased from Invitrogen Life calcium was monitored at emission of 526 nm with excitation of 496 nm Technologies (Carlsbad, CA). AA and human recombinant 5-LOX were using SpectraMax Gemini XS dual-scanning microplate spectrofluorome- from Cayman Chemicals (Ann Arbor, MI). The Abs for 5-LOX, ERK1/2 ter (Molecular Devices). (pT202/pY204), and ERK (pan ERK) were purchased from BD Bio- sciences Pharmingen (San Diego, CA). The secondary Abs were from Santa Cytoplasm membrane permeability Cruz Biotechnology (Santa Cruz, CA). Differentiated HL-60 cells were stimulated with A23187 in the presence or Isolation of human neutrophils absence of dT (50 mM). Cells were then washed with cold PBS buffer and stained with a kit containing annexin V–fluorescein and propidium iodide Neutrophils were isolated from fresh blood by density gradient centrifu- (PI) (Roche Diagnostics, Indianapolis, IN). The change of cytoplasmic gation using Histopaque-1077 (Sigma). Blood was obtained from healthy membrane permeability was monitored by Cell Lab Quanta SC–MPL flow donors following a protocol approved by human research protection pro- cytometer (Beckman Coulter), with excitation at 488 nm and emission at gram institutional review boards at Purdue University. 670 nm for PI. Cell differentiation Results HL-60 cells and clone 15 HL-60 cells were purchased from American Type Culture Collection (Manassas, VA). These cells were routinely maintained Vitamin E forms and 139-COOH inhibited A23187-stimulated in RPMI 1640 medium supplemented with 10% FBS under 5% CO . For 2 LTB4 in neutrophil-like differentiated HL-60 cells or human induction of neutrophil differentiation, 2.2 3 105 to 2.4 3 105 cells/ml neutrophils isolated from fresh blood of healthy subjects by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. were incubated in RPMI 1640 containing 10% FBS plus 1.25% DMSO for 5 d (18–20). For induction of eosinophil differentiation, clone 15 HL-60 Human promyeloblast HL-60 cells have been shown to be dif- cells were incubated in RPMI 1640 containing 10% FBS plus 0.5 mM ferentiated into neutrophils by 1.2–1.5% DMSO (18–20), and butyric acid for 5 d (21–23). lymphoblast clone 15 HL-60 cells are differentiated into eosino- Isolation of 139-COOH from cell culture media phils by butyric acid (21–23). To monitor the differentiation, we A549 cells were incubated with 50 mM dT for 72 h, and the media were measured the expression of 5-LOX, which only exists in mature collected for isolation of 139-COOH using a Supelcosil LC-18-DB Sem- granulocytes and is the key enzyme for leukotriene formation iprep column (Supelco, Bellefonte, PA) by HPLC as previously described (26). Under the culture conditions specified in Materials and (17). The isolated fractions were freeze-dried, quantified using HPLC, and Methods, 5-LOX expression reached maximum on the fourth to 2 http://classic.jimmunol.org stored at 80˚C until use. fifth day after the differentiation was initiated, at which time the Neutrophil and eosinophil activation differentiated cells can generate substantial amounts of leukotrienes; for example, when stimulated by A23187 (1 mM), dif- Neutrophils and differentiated HL-60 or clone 15 HL-60 cells (1.6 3 106) were preincubated with DMSO (0.05%), vitamin E forms, or 139-COOH in ferentiated HL-60 cells released 9.03–54.7 ng/ml LTB4 within 15 DMEM-1% FBS or HBSS (for experiments with S1P or LPA) at 37˚C for min compared with the baseline of 0.1–0.2 ng/ml. 10 min. Cells were then stimulated with A23187 or ionomycin (final 0.1% We found that vitamin E forms (Fig. 1) differentially inhibited DMSO) or other stimuli including S1P or LPA (final methanol at 1.9%) for Downloaded from A23187-stimulated LTB4 and LTC4 formation from differentiated another 15 min. After brief centrifugation, the medium was collected, and HL-60 cells and clone 15 HL-60 cells, respectively, and their LTB4 or LTC4 was measured by ELISA (Cayman Chemicals, Ann Arbor, MI). inhibitory potency depended upon the concentrations of A23187 (Fig. 2). When 1 mM A23187 was used for cell stimulation, gT, Western blotting dT, and gTE showed similar inhibitory potency with an IC50 of ∼5 To study 5-LOX translocation, differentiated cells were stimulated by mM (Fig. 2A,2B), whereas aT suppressed LTB4 and LTC4 at A23187 for 15 min, and the nuclear and cytosol fractions were isolated by IC50 of 60 and 40 mM, respectively. Similar effects were seen with a commercial kit, NE-PER nuclear and cytoplasmic extraction reagent ionomycin-stimulated leukotriene formation (data not shown). (Thermo Scientific, Waltham, MA). For ERK1/2 phosphorylation, differentiated cells were stimulated with A23187 for 2 min and were frozen When cells were stimulated by 2.5 mM A23187, the IC50 for gT immediately to stop cellular reactions. Collected cells were lysed in Tris- and dT increased to 20 mM (Fig. 2C). With A23187 at 5 mMor EDTA containing 1% SDS, 2 mM sodium vanadate, and protease inhibitor higher, none of the vitamin E forms showed consistent inhibition cocktails. The resulting solution was heated at 95˚C for 5 min. Proteins (25– even at 50 mM (data not shown). In contrast, 139-COOH, a long- 50 mg) were resolved on 10% precast SDS-PAGE gels, transferred onto a polyvinylidene floride membrane (Millipore, Billerica, MA), and probed chain metabolite from dT, dose-dependently inhibited LTB4 when by Abs. Membranes were exposed to chemiluminescent reagent (Perki- cells were stimulated by 1 or 5 mM A23187 with IC50 of 4 or 7 nElmer, Waltham, WA) and visualized on a Kodak film. mM (Fig. 2D), respectively. In addition, similar to the effects in The Journal of Immunology 1175

40% inhibition. In contrast, 139-COOH potently inhibited the enzyme activity with an IC50 of 0.5–1 mM (Fig. 3B), which is comparable with that of the speciﬁc 5-LOX inhibitor zileuton, which showed an IC50 of 3–5 mM. These results indicated that the re- duction of LTB4 in activated neutrophils by 139-COOH, but not the unmetabolized vitamins, likely stems from its direct inhibition of the 5-LOX activity. dT and gT inhibited A23187-stimulated 5-LOX translocation and ERK1/2 phosphorylation It is well established that in vivo activation of 5-LOX requires translocation of this enzyme from the cytosol to nuclear membrane, where it binds to 5-LOX activating protein and catalyzes leukotriene formation (19, 27–29). In the current study, we observed translocation of 5-LOX from cytosol to the nucleus in response to the stimulation of A23187 in the differentiated HL-60 cells. Consistent with the inhibition of LTB4, gT, dT, and gTE, but not aT, almost completely abolished A23187-induced 5-LOX translocation (Fig. 4A). Calcium-regulated signaling such as PKC and MEK-ERK has FIGURE 1. The structures of natural forms of vitamin E and 139-car- been shown to be important to the activation of 5-LOX translo- boxychromanol (139-COOH), a metabolite of dT. cation in granulocytes (4, 5, 30). Consistently, the speciﬁc inhibitor for PKC (Go6389) and MEK (U0126), but not that for p38 MAPK, potently inhibited A23187-induced LTB4 (Fig. 4B) and the differentiated cell lines, dT and 139-COOH potently suppres- LTC4 (data not shown). In agreement with the effect on 5-LOX sed LTB4 generation from ionophore-stimulated neutrophils iso- translocation, gT and dT impaired ionophore-triggered ERK1/2 lated from fresh blood of healthy subjects (Fig. 2E). phosphorylation (Fig. 4C), suggesting that the upstream signal- 2+ 139-COOH, but not vitamin E forms, potently inhibited human ing such as intracellular Ca increase may be modulated by vi- 5-LOX enzymatic activity tamin E forms. To understand the observed inhibition of leukotrienes by activated dT inhibited intracellular calcium increase stimulated by leukocytes, we investigated the effect of 139-COOH and vitamin E A23187 on human recombinant 5-LOX activity in a cell-free FOX assay Cytosolic calcium increase is the pivotal upstream event trigger- (24). In these assays, aT, gT, and dTat50mM did not exhibit any ing the activation of downstream signaling including PKC and effect on 5-LOX activity (Fig. 3A), whereas gT at 200 mM showed ERK1/2 phosphorylation that leads to 5-LOX translocation. Be- by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. http://classic.jimmunol.org Downloaded from

FIGURE 2. Vitamin E forms differentially inhibited A23187-induced LTB4 (A, C)orLTC4 (B) from neutrophil-like HL-60 cells or eosinophil-like clone 15 HL-60 cells, respectively. 139-COOH dose-dependently suppressed LTB4 production from A23187-stimulated (5 mM) HL-60 cells (D). dTand139-COOH inhibited LTB4 in stimulated human neutrophils isolated from fresh blood (E). Differentiated HL-60 and clone 15 HL-60 cells or freshly isolated neutrophils were preincubated with different vitamin E forms or 139-COOH for 10 min and then activated by A23187 for 15 min. LTB4 and LTC4 were measured by ELISA assays. The relative LTB4 (or LTC4) is the ratio of LTB4 (or LTC4) formed in the presence of tested compounds in relation to that with solvent control. The results are reported as mean 6 SD or SEM of two or more independent experiments. *p , 0.05; **p , 0.01 (difference between the treatment and solvent controls). 1176 VITAMIN E AND ITS LONG-CHAIN METABOLITE INHIBIT LEUKOTRIENES

FIGURE 3. The effect of vitamin E forms (A) and 139-COOH (B) on human recombinant 5-LOX activity. Human recombinant 5-LOX was preincubated with vitamin E forms and 139-COOH (at indicated concentrations, micromolar) or zileuton (10 mM) for 4 min at room temperature. Reactions were started by the addition of AA (ﬁnal 75 mM) and terminated by addition of FOX reagent. After color development, the absorbance was measured at 560 and 575 nm. The 5-LOX activity (%) is the ratio of the absorbance in the presence of tested compounds in relation to that of solvent control. *p , 0.05; **p , 0.01 (difference between the treatment and solvent controls).

cause tocopherols inhibited 5-LOX translocation and ERK1/2 protein-coupled, and phospholipase C-involved mechanism (7). phosphorylation, we reason that they may modulate intracellular dT treatment did not affect LTB4 induced by THAP (data not calcium increase. As expected, we found that dT greatly sup- shown), which depletes ER Ca2+ storage by noncompetitively in- pressed A23187-stimulated cytosolic calcium increase (Fig. 5A). hibiting ER Ca2+ ATPase (31). In contrast, dT potently suppressed In addition, dT appeared to block calcium influx when extracel- LTB4 stimulated by S1P and LPA (Fig. 6A), which have been lular calcium was added (Fig. 5B). shown to stimulate calcium influx via a receptor-independent mechanism (8, 9). Consistent with the effect on S1P-mediated dT inhibited LTB generation stimulated by S1P or LPA, but 4 LTB , dT also inhibited S1P-triggered calcium increase and cal- not by THAP or fMLP, and blocked S1P-stimulated 4 cium influx (Fig. 6B,6C). Unlike dT, its metabolite 139-COOH at intracellular calcium increase, whereas the inhibitory effects low micromolar concentration strongly inhibited fMLP- and by 139-COOH were independent of stimulus types THAP-stimulated LTB4 (data not shown). To understand the mechanisms underlying the inhibition of calcium dT counteracted ionophore-enhanced membrane permeability increase, we tested the potential effect of dTonLTB4 generation induced by several stimuli that are known to increase cytosolic Because dT suppressed LTB4 production stimulated by S1P, LPA, calcium via distinct mechanisms (Fig. 6A). dT showed no effects or ionophores that are known to increase intracellular Ca2+ by on LTB4 formation induced by fMLP, which causes calcium re- distinct mechanisms (8, 9), we reason that dT may modulate mem- lease from intracellular ER storage via a receptor-mediated, G brane perturbation induced by these stimuli. Therefore, we used PI by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd.

FIGURE 4. A, Effects of vitamin E forms on A23187-induced translocation of 5-LOX. Differenti- ated HL-60 cells were treated with 50 mM of different forms of vitamin E or solvent controls for 10 min and then stimulated by A23187 (2.5 mM) for 15 min. 5- http://classic.jimmunol.org LOX in the cytosolic and nuclear fractions were probed by Western blotting. B, The effect of speciﬁc inhibitors

for various signaling pathways on LTB4 formation. Differentiated HL-60 cells were preincubated with inhibitors of MEK/ERK (U0126 at 20 mM, bar 3), PKC (Go-6983 at 2 or 10 mM, bars 4 and 5), or MAPK p38 (SB202190 at 5 or 10 mM, bars 6 and 7) for 10 min and Downloaded from then stimulated by A23187 (5 mM) for 15 min. Bars 1 and 2 are vehicle controls without or with A23187, respectively. *p , 0.05; **p , 0.01 (difference between cells treated with tested compounds and solvent control [bar 2]). C, gT and dT inhibited A23187-induced phosphorylation of ERK1/2 in neutrophils. Dif- ferentiated HL-60 cells were treated with or without dT or gT (50 mM) for 10 min and then stimulated by 1 mM A23187 for 2 min. Phosphorylated ERK1/2 and whole ERK1/2 were probed by Western blotting. The Journal of Immunology 1177

only slightly attenuated 10 mM ionophore caused calcium release (Fig. 7). These data were consistent with the observation that when relatively high concentrations (.5 mM) of A23187 were used for cell stimulation, vitamin E forms failed to reduce LTB4 production. These results suggest that dT may block calcium entry via preventing membrane changes induced by the stimuli.

Discussion LTB4 produced by 5-LOX–catalyzed reaction is one of the most potent chemotactic agents for leukocytes (1, 32) and thus plays a significant role in regulation of inflammatory response. Cys- FIGURE 5. A, The effect of dT on intracellular calcium increase induced teinyl leukotrienes such as LTC4 that are generated by eosino- by A23187. Differentiated HL-60 cells were incubated with 2 mM fluo-4 phils or mast cells are known to contribute to the development of AM in HBSS in the dark for 30 min. After a brief washing, cells were 2+ asthma and allergic inflammation (1, 33, 34). Leukotriene antag- resuspended in HBSS buffer with Ca and dT (50 mM) or 0.05% DMSO onists and 5-LOX inhibitors have been used to treat asthma and (solvent control) at 37˚C for 10 min and then stimulated with 0.5 or 1 mM inflammatory diseases (2). Here we have demonstrated that gT, A23187. Symbols: triangles are calcium increase with A23187 at 1 mM (open, solvent; solid, dT), and circles are that with A23187 at 0.5 mM dT, and gTE, at physiologically relevant concentrations, suppres- (open, solvent; solid, dT). B, The effect of dT on A23187-mediated Ca2+ sed neutrophil-mediated LTB4 production much more potently influx. After preloading with fluo-4 AM, cells were incubated with dT (50 than aT by blocking intracellular calcium increase, which con- mM) for 10 min in HBSS without Ca2+. Cells were stimulated by A23187 sequently led to inhibition of ERK phosphorylation and 5-LOX (0.5 mM) for 1 min 20 s, and then Ca2+ added (final 1.25 mM). Symbols: translocation, a key step for activation of 5-LOX. Unlike the un- open and solid circles represent calcium increase in vehicle controls and metabolized vitamins, 139-COOH, a long-chain metabolite of vi- dT-treated cells, respectively. In all these studies, intracellular calcium was tamin E, decreased leukotriene formation by direct inhibition of monitored by measuring fluo-4 AM at emission of 526 nm with excitation the 5-LOX activity. of 494 nm. Calcium increase is the subtraction of fluorescence intensity in We have previously demonstrated that 139-COOH derived from the presence of A23187 minus fluorescence intensity without the stimulus. dT strongly inhibits COX-1 and COX-2 by competing with AA for the binding to the substrate site of COXs (17). In this study, we to monitor potential cytoplasmic membrane disruption in response found that 139-COOH inhibits 5-LOX activity at a potency com- to the stimulation. Two minutes after cells were treated with parable with that of zileuton, a competitive inhibitor of 5-LOX. A23187, the cytoplasmic membrane integrity was not affected Consistent with its strong inhibition of 5-LOX activity, 139-COOH (Fig. 7). In contrast, membrane permeability appeared to be en- suppressed cellular production of LTB4 regardless of the stimuli. hanced during the extended 4-min incubation. dT completely re- Given that both 139-COOH and AA share an end carboxylate versed the enhanced PI staining when A23187 was at 1 mM, and group and both bind to the substrate site of COXs, we postulate that 139-COOH may also competitively inhibit human 5-LOX,

by guest on October 1, 2021. Copyright 2010 Pageant Media Ltd. which warrants further investigation. Regardless of the mechanisms, the unique dual inhibition of COXs and 5-LOX by 139- COOH makes it an interesting and novel anti-inflammatory agent. This is because compared with specific COX or 5-LOX inhibitors, 139-COOH may exhibit more potent anti-inflammatory effects by suppressing multiple proinflammatory pathways and may have reduced adverse effects caused by a shunt of AA metabolism to either the COX- or 5-LOX–mediated pathway (35, 36). In contrast with their long-chain metabolite, vitamin E forms http://classic.jimmunol.org failed to affect human recombinant 5-LOX activity at up to 50 mM, although aT has previously been reported to noncompetitively Downloaded from

FIGURE 6. A, Effects of dTonLTB4 production stimulated by fMLP, S1P, and LPA from differentiated HL-60 cells. Cells were preincubated with dT (50 mM) for 10 min and stimulated by fMLP (1 mM), S1P (50 FIGURE 7. Effects of dT on A23187-enhanced membrane permeability.

mM), or LPA (100 mM) for 10 min. LTB4 was measured by ELISA. *p , Differentiated HL-60 cells were preincubated with dT (50 mM) for 10 min 0.05; **p , 0.01 (difference between dT treatment and solvent controls for and then stimulated with A23187 (1 or 10 mM) for 2 or 4 min. The each indicated activator). B and C, Effects of dTon(B) S1P-triggered membrane permeability was measured by PI staining using ﬂow cytometry. intracellular calcium increase and (C) calcium inﬂux. The experimental *p , 0.02 (difference between A1 and dT + A1); *p , 0.05 (difference procedure is the same as that described for Fig. 5; concentration of S1P between A10 and dT + A10). A1, A23187 at 1 mM; A10, A23187 at 10 was 50 mM. mM; Ctrl, no stimulus. 1178 VITAMIN E AND ITS LONG-CHAIN METABOLITE INHIBIT LEUKOTRIENES

inhibit potato 5-LOX at much lowered concentrations (37). This vitamin E levels (10, 12, 14), whereas high aT(.100 mM) discrepancy of vitamin E toward human and potato 5-LOX is appears to be effective to suppress 5-LOX products (current study likely due to the large sequence difference between the two en- and Refs. 10, 47). zymes (based on Blast protein sequence comparison). However, Unlike aT, the strong inhibition of leukotrienes by other vita- the lack of inhibition of human 5-LOX activity is in agreement min E forms such as gT and their long-chain metabolites likely, at with the fact that the inhibitory effect of vitamin E forms on least in part, account for the in vivo anti-inflammatory activities cellular LTB4 formation varies with the specific stimuli, that is, that have been reported by us and others. This is because plasma showing inhibition to A23187, S1P, and LPA but not fMLP or and liver concentrations of gT have been reported to reach up to THAP stimulation, even when comparable amounts of LTB4 were 20 or 60 mM (48, 49), respectively, and 139-COOH is detected in generated (Fig. 6A). Our results are also consistent with the pre- tissues in response to vitamin E supplementation (48, 50). Thus, vious study by Goetzel (10) showing that aTat,30 mM enhances we have shown that gT but not aT potently inhibits carrageenan- 5-LOX–catalyzed products by A23187-stimulated human neu- induced LTB4 at the inflammation site in the rat air-pouch model trophils, whereas a suppressive effect is observed at .120 mM (14), which is consistent with the current observation that gT more (10). Notably, we also observed a slight enhancement of leukotriene potently suppressed cellular leukotriene formation than that by by low micromolar concentration of aT(Fig.2),andaT supple- aT. gT supplementation also alleviates OVA-sensitized airway in- mentation at 33 mg/kg body weight tends to increase inflammation- flammation and suppresses leukotriene increase in the broncho- induced LTB4 in a rat inflammation model (14). alveolar lavage fluid in Brown Norway rats (15, 16). In addition, It is intriguing that vitamin E forms suppress LTB4 by modu- gT-rich mixed tocopherols lower LTB4 in an inflammation- lation of calcium influx and that their effect is dependent upon the promoted colon carcinogenesis model (51). Our current findings types of activator. Because dT failed to affect fMLP- or THAP- also suggest that dTandgTE, as well as long-chain carboxy- stimulated leukotriene, vitamin E forms do not likely have impact chromanols, appear to have equal or stronger anti-inflammatory on fMLP receptor-mediated, phospholipase C-involved signaling, effects compared with those of gT. Future studies should be car- or Ca2+ store depletion. The lack of influence on the receptor- ried out in animals and humans to further determine the role of mediated pathway is also supported by the strong inhibition of supplementation of these vitamin E forms and their long-chain LTB4 stimulated by S1P and LPA, which increase intracellular metabolites in inflammation-related diseases. calcium via receptor-independent mechanisms in human neutrophils and HL-60 cells (8, 9). Although S1P and LPA appear to Disclosures trigger store-operated and non-store-operated calcium entry, re- The authors have no financial conflicts of interest. spectively (8, 9), the exact mechanisms leading to calcium influx is unknown. In contrast, because the ionophore appears to enhance membrane permeability as indicated by the increase of intra- References cellular PI staining, we propose that dT blocks calcium influx by 1. Funk, C. D. 2001. Prostaglandins and leukotrienes: advances in eicosanoid bi- counteracting stimuli-caused disruption of cytoplasmic membrane ology. Science (New York, N. Y.) 294: 1871–1875. . 2. Peters-Golden, M., and W. R. Henderson, Jr. 2007. Leukotrienes. N. Engl. J. integrity. However, when higher concentrations of ionophore ( 5 Med. 357: 1841–1854.

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