Inhibition of Neutrophil Leukotriene B4 Production by a Novel Synthetic N-3 Polyunsaturated Analogue, β-Oxa 21:3 n-3 This information is current as of September 25, 2021. Brenton S. Robinson, Deborah A. Rathjen, Neil A. Trout, Christopher J. Easton and Antonio Ferrante J Immunol 2003; 171:4773-4779; ; doi: 10.4049/jimmunol.171.9.4773 http://www.jimmunol.org/content/171/9/4773 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Inhibition of Neutrophil Leukotriene B4 Production by a Novel Synthetic N-3 Polyunsaturated Fatty Acid Analogue, ␤-Oxa 21:3n-31

Brenton S. Robinson,* Deborah A. Rathjen,2*ʈ Neil A. Trout,*§ Christopher J. Easton,¶ and Antonio Ferrante3*†‡

We recently reported the synthesis and anti-inflammatory properties of a novel long chain polyunsaturated fatty acid (PUFA) with an oxygen atom in the ␤-position, ␤-oxa-21:3 n-3 (Z,Z,Z)-(octadeca-9,12,15-trienyloxy) ). Our data, from studies aimed at elucidating the mechanism of its action, show that pretreatment of human neutrophils with the ␤-oxa-PUFA substantially depresses the production of leukotriene B4 (LTB4) in response to calcium ionophore, A23187, comparable to standard leukotriene inhibitors such as zileuton and nordihydroguaiaretic acid. Interestingly, the n-6 equivalent, ␤-oxa 21:3 n-6, is also a strong Downloaded from inhibitor of LTB4 production. In contrast, naturally occurring PUFA only slightly reduce, for eicosapentaenoic (20:5n-3) and ␤ docosahexaenoic (22:6n-3) acids, or increase, for (20:4n-6), the formation of LTB4. The parent -oxa-21:3n-3 molecule, rather than its derivatives (methyl ester, saturated, monohydroperoxy, or monohydroxy forms), is exclusively respon- ␤ 3 3 sible for attenuation of LTB4 formation. -Oxa-21:3n-3 inhibits the conversion of [ H]20:4n-6 to [ H]5-hydroxyeicosatetraenoic 3 acid and [ H]LTB4 by neutrophils in the presence of calcium ionophore and also suppresses the activity of purified 5-lipoxygenase, but not cyclooxygenase 1 and 2. ␤-Oxa-21:3n-3 is taken up by neutrophils and incorporated into phospholipids and neutral . http://www.jimmunol.org/ In the presence of calcium ionophore, the leukocytes convert a marginal amount of ␤-oxa-21:3n-3 to a 16-monohydroxy-␤-oxa- 21:3n-3 derivative. After administration to rodents by gavage or i.p. injection, ␤-oxa-21:3n-3 is found to be incorporated into the lipids of various tissues. Thus, ␤-oxa-21:3n-3 has the potential to be used in the treatment of inflammatory diseases, which are mediated by products of the lipoxygenase pathway. The Journal of Immunology, 2003, 171: 4773–4779.

olyunsaturated fatty acids (PUFA)4 are ubiquitous compo- idation (6). Apart from their important role as structural compo- nents of mammalian cell membranes, where they are gen- nents and as a source of energy, some of these fatty acids, in erally esterified in phospholipids and neutral lipids (1). In particular eicosatetraenoic acid (arachidonic acid, 20:4n-6) and ei- P by guest on September 25, 2021 addition to membranes, another PUFA-bearing domain in cells can cosapentaenoic acid (20:5n-3), act as precursors of physiologically be bodies, which are lipid-rich cytoplasmic inclusions (2Ð4). active oxygenated metabolites, collectively termed eicosanoids. Most cellular PUFA are derived from the essential dietary fatty These include prostaglandins, thromboxanes, prostacyclins (cyclo- acids, octadecadienoic acid (, 18:2n-6) and octadeca- oxygenase products), hydroxylated fatty acids, leukotrienes, li- trienoic acid (␣-linoleic acid, 18:3n-3) by the process of chain poxins (lipoxygenase products), and epoxygenated fatty acids (ep- elongation and desaturation (5) and are mainly degraded by ␤-ox- oxygenase products) (7Ð11). When leukocytes such as neutrophils are challenged with certain agonists, the formation of leukotrienes is initiated by the release of *Department of Immunopathology, Women’s and Children’s Hospital, North Ad- 20:4n-6 from phospholipids by the activation of phospholipase A2. elaide, South Australia, Australia; and †Department of Pediatrics, University of Ad- elaide, South Australia, Australia; ‡School of Pharmaceutical, Molecular and Bio- The unesterified 20:4n-6 then undergoes 5-lipoxygenation to 5-hy- sciences, University of South Australia, Australia; ¤Department of Chemistry, droperoxyeicosatetraenoic acid (5-HPETE), which is further con- Flinders University of South Australia, Bedford Park, South Australia, Australia; verted to an unstable epoxide leukotriene A (LTA ). This inter- ¶Research School of Chemistry, Australian National University, Canberra, Australian 4 4 ʈ Capital Territory, Australia; and Peptech Ltd., North Ryde, New South Wales, Aus- mediate can be enzymatically hydrolyzed to LTB4. Alternatively, tralia LTA4 is transformed, by the addition of glutathione at C-6, into Received for publication April 4, 2003. Accepted for publication August 12, 2003. leukotriene C4 (LTC4). Stepwise enzymatic elimination of glu- The costs of publication of this article were defrayed in part by the payment of page tamic acid and glycine in the peptide side chain leads to formation charges. This article must therefore be hereby marked advertisement in accordance of LTD and LTE , respectively (9Ð11). The presumed asthma with 18 U.S.C. Section 1734 solely to indicate this fact. 4 4 mediator slow-reacting substance of anaphylaxis has now been 1 This work was supported by the National Health and Medical Research Council of Australia, the National Heart Foundation of Australia, and the Adelaide Women’s and identified as a biological activity composed of LTC4, LTD4, and Children’s Hospital Research Foundation. LTE4 (cysteinyl-leukotrienes), which are all potent bronchocon- 2 Current addresses: Bionomics Ltd., 31 Dalgleish Street, Thebarton, South Australia strictors (12Ð14). In addition, these leukotrienes have been impli- 5031, Australia. cated in hypoxic vasoconstriction and the pathophysiology of re- 3 Address correspondence and reprint requests to Prof. A. Ferrante, Department of Im- spiratory distress syndromes (15). The closely related compound, munopathology, Women’s and Children’s Hospital, 72 King William Road, North Ad- elaide, South Australia 5006, Australia. E-mail address: [email protected] LTB4, is a potent mediator of leukocyte-dependent inflammation 4 Abbreviations used in this paper: PUFA, polyunsaturated fatty acid(s); DPC, di- (16, 17) and hence is important for the inflammatory component of palmitoylphosphatidylcholine; GLC, gas-liquid chromatography; LTB4, leukotriene asthma and other pulmonary afflictions. B4; MS, mass spectrometry; 20:4n-6, eicosatetraenoic acid (arachidonic acid); 22: 6n-3, ; 20:5n-3, ; NDGA, nordihydroguai- Our recent studies have shown that PUFA also have the ability aretic acid. to directly stimulate leukocytes (neutrophils and macrophages) to

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 ␤ 4774 INHIBITION OF NEUTROPHIL LTB4 PRODUCTION BY A -OXA PUFA release superoxide, degranulate, adhere, and display increased ex- HBSS by sonication (19). Controls contained micelles of DPC alone sim- pression of CR3 receptors (18Ð20). We have observed that the ilarly diluted. TLC and gas-liquid chromatography (GLC)-mass spectrom- modulation of biological activity induced by PUFA is related to etry (MS) indicated that the lipids were of high purity. Agonists and media were shown to be free of endotoxin contamination using the Limulus ame- their structure (21). These effects of naturally occurring PUFA on bocyte lysate assay. In the TLC analysis the oxa fatty acid is UV active, neutrophils are clearly proinflammatory and therefore would ex- such that when the TLC plate is sprayed with dichlorofluorescein and acerbate the asthmatic state. viewed under UV light, it quenches the fluorescence of the dichlorofluo- 20:5n-3 and docosahexaenoic acid (22:6n-3), which are prom- rescein to show up as a dark purple spot on a green background. inent in diets enriched with fish oil, competitively inhibit the con- Isolation of neutrophils version of 20:4n-6 to eicosanoids. With respect to leukotriene Human neutrophils were isolated from the peripheral blood of healthy vol- products of the 5-lipoxygenase pathway in leukocytes, LTB5, the unteers by the rapid single-step method described by Ferrante and Thong dihydroxy derivative of 20:5n-3 formed from LTA5, is a weak and (32) in which the blood was layered onto Ficoll-Hypaque medium (density, ϫ partial agonist compared with LTB4 in eliciting inflammatory re- 1.114 g/L) and centrifuged at 600 g for 30Ð40 min at room temperature. sponses (11, 22, 23). The pentaenoic cysteinyl-leukotrienes are The leukocytes resolved into two distinct bands, with the lower containing equiactive with their tetraenoic counterparts (11, 24, 25). Dietary neutrophils. In the majority of cases the preparation of neutrophils was of Ͼ99% purity and Ͼ99% viability, as judged by morphological examina- strategies involving fish oils rich in 20:5n-3 and 22:6n-3 for in- tion of cytospin preparations and the ability of viable cells to exclude creasing the amount of LTB5 production at the expense of LTB4 trypan blue stain. The neutrophils were washed three times, resuspended in production have attracted interest because of their potential use in HBSS (5 ϫ 106 cells/ml), and used within 30 min of preparation. Trypan the management of a range of human diseases, including asthma, blue exclusion and lack of lactate dehydrogenase release indicated that the cells remained viable throughout subsequent incubations. rheumatoid arthritis, systemic lupus erythematosis, and cardiovas- Downloaded from cular diseases (26). However, to date the results of such studies Measurement of LTB4 production have essentially remained controversial and disappointing with re- Neutrophils (5 ϫ 106) were preincubated with or without fatty acids or spect to their use in the treatment of inflammatory diseases (27). derivatives (2.5Ð40 ␮M), Zileuton (10 ␮M; 5-lipoxygenase inhibitor) or This is probably due to 20:5n-3 and 22:6n-3 having direct proin- NDGA (10 ␮M, 5-, 12-, and 15-lipoxygenase inhibitor) in 0.5 ml of HBSS flammatory effects, such as stimulation of leukocyte respiratory at 37¡C for up to 60 min. The cells were subsequently incubated in the presence or the absence of the calcium ionophore A23187 (5 ␮M) in 1 ml burst, degranulation, and adherence. final volume of HBSS at 37¡C for 30 min. The incubates were immediately http://www.jimmunol.org/ Recently we described a chemically synthesized novel PUFA, centrifuged at 14,000 ϫ g at 4¡C for 5 min, and the supernatants were ␤ ␤ Ϫ with an oxygen atom in the -position, -oxa-21:3n-3 (28), that stored at 70¡C until analysis. The amount of LTB4 in the medium sam- has immunosuppressive properties, but, unlike its natural PUFA ples was measured using commercial enzyme immunoassay kits. counterpart, was a weak stimulator of neutrophil functions (29). In Determination of [3H]5-hydroxyeicosatetraenoic acid ([3H]5- the present study we have assessed whether such chemically en- 3 3 HETE) and [ H]LTB4 production from [ H]20:4n-6 gineered PUFA inhibit calcium ionophore-induced production of The production of [3H]5-HETE and [3H]LTB by [3H]20:4n-6-prelabeled LTB by human neutrophils. 4 4 neutrophils challenged with the calcium ionophore A23187 was assessed as follows. Neutrophils (6 ϫ 107) were incubated with [3H]20:4n-6 (10 Materials and Methods ␮Ci; 24 nM) in 2 ml of HBSS at 37¡C for 75 min. The cells were washed by guest on September 25, 2021 Fatty acids three times with 10 ml of HBSS containing BSA (0.001%, w/v), once with 10 ml of HBSS, and finally resuspended in HBSS. Approximately 80% of ␤-Oxa-PUFA, ␤-oxa-21:3n-3, and ␤-oxa-21:3n-6, were synthesized as de- the radiolabeled fatty acid substrate was taken up by the cells, of which scribed by Pitt et al. (28). ␤-Oxa-21:3n-3 methyl ester was formed by Ͼ98% was esterified in lipids. [3H]20:4n-6-labeled neutrophils (2 ϫ 107) treatment of ␤-oxa-21:3n-3 with diazomethane in diethyl ether, and ␤-oxa- were preincubated with or without ␤-oxa-21:3n-3 (20 ␮M)in2mlof 21:0 was prepared by hydrogenation of ␤-oxa-21:3n-3 in the presence of HBSS at 37¡C for 15 min. The cells were then incubated in a 4-ml final platinum oxide (30). 16-Monohydroperoxy-␤-oxa-21:3n-3 was prepared volume of HBSS containing the calcium ionophore A23187 (5 ␮M) at by incubation of ␤-oxa-21:3n-3 with soybean lipoxidase (31), and 16-mo- 37¡C for up to 30 min. Lipids were extracted by the addition of 15 ml of nohydroxy-␤-oxa-21:3n-3 was obtained by reduction of the 16-monohy- chloroform/methanol/acetic acid (1/2/0.02, v/v/v) (33). The mixture was droperoxy product with sodium borohydride (31). 20:4n-6, docosahexae- left at room temperature for 1 h and subsequently partitioned by the ad- noic acid (22:6n-3), eicosapentaenoic acid (20:5n-3), and nonadecanoic dition of 5 ml of chloroform and 5 ml of water (34). Concentrated lipid acid (nonadecylic acid, 19:0) methyl ester were obtained from Sigma-Al- extracts were applied to silica gel 60 TLC plates and developed in diethyl drich (St. Louis, MO). ether/hexane/acetic acid (60/40/1, v/v/v) or the organic phase of ethyl ac- etate/iso-octane/acetic acid/water (11/5/2/10, v/v/v/v) to isolate [3H]5- Biochemical and inhibitors 3 HETE and [ H]LTB4 (35, 36). Identification of the lipids was based on a The calcium ionophore A23187, nordihydroguaiaretic acid (NDGA), di- comparison of their TLC mobility with that of authentic unlabeled stan- palmitoylphosphatidylcholine (DPC), BSA (essentially fatty acid free), and dards. The 5-HETE and LTB4 zones were located with I2 vapor or by butylated hydroxytoluene were purchased from Sigma-Aldrich. preparing autoradiographs and were scraped into scintillation vials, and the [5,6,8,9,11,12,14,15-[3H]20:4n-6 (211 Ci/mmol) and high performance au- radioactivity was measured after adding water (0.5 ml) and scintillation toradiography film (Hyperfilm-3H) were purchased from Amersham Aus- fluid (10 ml) with a liquid scintillation counter (model 1409 set with ex- tralia (North Ryde, Australia), and Zileuton was supplied by Abbott Lab- ternal standardization and automatic efficiency control to correct for oratories (North Chicago, IL). Silica gel 60 TLC plates (20 cm ϫ 20 cm ϫ quenching; LKB Wallac). 3 3 0.25 mm) were obtained from Merck (Darmstadt, Germany), scintillation In other experiments the production of [ H]5-HETE and [ H]LTB4 by 3 cocktail (Opti Phase Hi Safe 3) was purchased from LKB Wallac (Turku, neutrophils treated simultaneously with [ H]20:4n-6 and the calcium iono- phore A23187 was examined. Neutrophils (2 ϫ 107) were preincubated Finland), and LTB4 enzyme immunometric assay kits were purchased from Cayman Chemical (Ann Arbor, MI). All other chemicals were of reagent with or without ␤-oxa-21:3n-3 (20 ␮M) in 2 ml of HBSS at 37¡C for 15 grade. Solvents were distilled before use and contained the antioxidant min. The cells were then incubated in a 4-ml final volume of HBSS con- 3 butylated hydroxytoluene (0.005%, w/v). taining [ H]20:4n-6 (2 ␮Ci, 5 ␮M) and calcium ionophore A23187 (5 ␮M) at 37¡C for up to 30 min. The radioactivity associated with 5-HETE and

Presentation of fatty acids and agonists LTB4 was determined as described above.

Fatty acids and derivatives, Zileuton, NDGA, and calcium ionophore Assessment of 5-lipoxygenase, LTA4 hydrolase, and A23187 (concentrated stocks in ethanol stored at Ϫ20¡C) were diluted with cyclooxygenase activity HBSS immediately before use. Ethanol alone appropriately diluted in HBSS was used as a control. The final concentration of ethanol in neutro- The effects of ␤-oxa Ϫ21:3n-3 on purified 5-lypoxygenase, cyclooxygen- Ͻ phil incubations was 0.2% (v/v). In some cases fatty acids were also ase 1 and 2, and LTA4 hydrolase were determined by PanLabs (Bothwell, solubilized by preparing mixed DPC/fatty acid micelles (4/1, w/w) in WA). Cyclooxygenase 1 activity was measured using the enzyme purified The Journal of Immunology 4775

from ram seminal vesicle using radiolabeled 20:4n-6 substrate at 37¡C for recovered oxygenated fatty acid derivatives were taken up in methanol and 20 min (37). Cyclooxygenase 2 activity was similarly measured using the characterized by electrospray MS according to the method described by

enzyme purified from sheep placenta (38). LTA4 hydrolase activity was Pitt et al. (43). determined by incubating the enzyme purified from guinea pig lungs and

radiolabeled LTA4 substrate incubated at 25¡C for 4 min (39). The effects Statistical analyses on 5-lipoxygenase activity was determined using the enzyme purified from Ϯ rat RBL-1 cells and radiolabeled 20:4n-6 as substrate incubated at 25¡C for Results are expressed as the mean SEM. Statistical analyses were per- formed by one-way ANOVA, followed by Dunnett’s test for multiple com- 8 min (40) Ͻ These studies were conducted under standard operating procedures de- parisons or by two-tailed Student’s t test for unpaired data. A value of p veloped by Panlabs. Concurrent testing of standard reference compounds 0.05 was considered significant. (indomethacin at 1.7 ␮M for cyclooxygenase 1; 2.4 ␮M for cyclooxygen- ␮ ␮ ase 2, 49 M ebselen for LTA4 hydrolase; 0.25 M NDGA) was per- Results formed with each protocol. Inhibition of 5-HETE and LTB4 production Assessment of ␤-oxa-21:3n-3 incorporation into cellular lipids Pretreatment of human neutrophils with engineered ␤-oxa-PUFA ␤ ␤ Neutrophils (4 ϫ 107) were incubated with or without 20 ␮M ␤-oxa-21: ( -oxa-21:3n-3, -oxa-21:3n-6) markedly inhibited the generation 3n-3 in 4 ml of HBSS at 37¡C for up to 60 min. The incubate was cen- of LTB4 when the cells were stimulated with the calcium iono- trifuged at 600 ϫ g for 5 min at room temperature, and the medium was phore A23187 (Fig. 1). This effect was comparable to that ob- discarded. The cell pellet was resuspended in 2 ml of water, and lipids were served with the commercially available leukotriene inhibitors, extracted by the addition of 7.5 ml of chloroform/methanol/acetic acid Zileuton and NDGA. In contrast, pretreatment with naturally oc- (1/2/0.02, v/v/v) (33). The mixture was left at 4¡C overnight and then partitioned by the addition of 2.5 ml of chloroform and 2.5 ml of water curring PUFA only slightly suppressed, in the case of 20:5n-3 and (34). 22:6n-3, or increased, in the case of 20:4n-6, the ionophore-in- Downloaded from The amount of ␤-oxa-21:3n-3 associated with total cellular lipid was duced LTB4 production (Fig. 1). The fatty acids were presented to measured as follows. Total lipid extracts were evaporated to dryness under the leukocytes with ethanol as diluent (0.1%, v/v, final concentra- N2 and transesterified with 5 ml of 1.5% (v/v) H2SO4 in methanol at 75¡C for 4 h. The resulting fatty acid methyl esters were extracted with 1 ml of tion); however, similar results were observed using mixed fatty water and 2 ml of hexane and then purified on silica gel 60 TLC plates, acid-DPC micelles. Trypan blue exclusion and lack of lactate de- which were developed in dichloromethane. Authentic standards were co- hydrogenase release showed that the cells remained viable under chromatographed to assist identification of the methyl ester species. The our experimental conditions (data not presented). The inhibitory http://www.jimmunol.org/ ␤ -oxa-fatty acid methyl ester zone was located under UV light after spray- effect of ␤-oxa-21:3n-3 on LTB production was independent of ing the plates with 0.1% (w/v) dichlorofluorescein in ethanol and eluted 4 from the silica gel with two 4-ml volumes of chloroform/methanol/acetic pretreatment time (Fig. 2A) and was significant with a concentra- acid/water (50/39/1/10, v/v/v/v) (41). The resulting extracts were parti- tion of Ն5 ␮M (Fig. 2B) tioned with 2 ml of1MNH4OH to remove the dye and then washed with Further studies were conducted to determine whether the gen- ␤ 2 ml of methanol/water (1/1, v/v) (37). -Oxa-21:3n-3 methyl ester was eration of [3H]5-HETE was affected by ␤-oxa-21:3n-3. Pretreat- quantitated by GLC using 19/0 methyl ester (48 nmol) as a reference stan- 3 ␤ dard (42). For more definitive identification of the fatty acid methyl esters, ment of [ H]20:4n-6-prelabeled neutrophils with -oxa-21:3n-3 a combined GLC-MS technique was employed (42). significantly inhibited calcium ionophore-enhanced production of ␤ 3 3 The amount of -oxa-21:3n-3 associated with various lipid classes was [ H]5-HETE (Fig. 3A) and [ H]LTB4 (Fig. 3B). The high 5-HETE by guest on September 25, 2021 determined as follows. Concentrated lipid extracts were applied to silica and LTB seen at time zero is probably a result of some neutrophil gel 60 TLC plates and developed in the first dimension in chloroform/ 4 methanol/ammonia (65/25/5, v/v/v) and then in the second dimension in chloroform/acetone/methanol/acetic acid/water (40/30/10/10/5, v/v/v/v/v) to separate phospholipids. Concentrated lipid extracts were applied to silica gel 60 TLC plates and developed in hexane/diethyl ether/acetic acid (80/ 20/1, v/v/v) to resolve neutral lipids. Identification of the lipids was based on a comparison of their TLC mobility with that of authentic standards. The lipid zones were located under UV light after spraying the plates with dichlorofluorescein and were eluted from the silica gel. The oxa fatty acid is UV active, so on a TLC plate sprayed with the dichlorofluorescein and viewed under UV light, it quenches the fluorescence of the dichlorofluo- rescein to show up as a dark spot on a green background. The samples were transesterified, and the liberated fatty acid methyl esters were quantitated by GLC as described above. The positional distribution of ␤-oxa-21:3n-3 in purified phosphatidylcholine, phosphatidylethanolamine, and phospha- tidylinositol (major neutrophil phospholipids) was assessed by degrading

the compounds with phospholipase A2 and measuring the amount of the ␤-oxa-PUFA associated with the reaction products (unesterified fatty acids and lysophospholipids) after isolation by TLC (42). Studies of the conversion of ␤-oxa-21:3n-3 to oxygenated fatty acid derivatives ␤ Neutrophils (2 ϫ 107) were treated with or without ␤-oxa-21:3n-3 (20 ␮M) FIGURE 1. Effect of -oxa and natural PUFA on LTB4 production by in 2 ml of HBSS at 37¡C for 15 min and then incubated with the calcium neutrophils challenged with the calcium ionophore A23187. Neutrophils ionophore A23187 (5 ␮M) in a 4-ml final volume of HBSS at 37¡C for an (5 ϫ 106) were preincubated with PUFA (20 ␮M), Zileuton (10 ␮M), additional 30 min. In some experiments the cells were preincubated with NDGA (10 ␮M), or diluent (control) in 0.5 ml of HBSS at 37¡C for 15 min. NDGA (10 ␮M) or diluent (control) in 1 ml of HBSS at 37¡C for 5 min. The cells were then incubated ina1mlfinal volume of HBSS containing The incubate was centrifuged, and lipids were extracted from the medium calcium ionophore A23187 (5 ␮M) at 37¡C for 30 min. The amount of as described above for the lipid incorporation studies (33, 34). The con- LTB4 in the medium was determined as described in Materials and Meth- centrated lipid extract (pooled from 10 samples) was applied to a silica gel ods. Each bar represents the mean Ϯ SEM for three determinations. This 60 TLC plate and developed in diethyl ether/hexane/acetic acid (60/40/1, p Ͻ 0.01 (for ,ء .experiment was performed three times with similar results v/v/v) to resolve oxygenated fatty acid derivatives. Authentic standards were cochromatographed to assist identification of the fatty acid species. significant differences between pretreatment with compounds and control, The lipid areas were located under UV light after spraying the plate with by one-way ANOVA, followed by Dunnett’s test for multiple compari- 0.01% (w/v) primulin in acetone/water (4/1, v/v) and were eluted from the sons). The basal production of LTB4 by the cells was negligible and was silica gel with three 4-ml volumes of chloroform/methanol (2/1, v/v). The not significantly affected by agonist pretreatment. ␤ 4776 INHIBITION OF NEUTROPHIL LTB4 PRODUCTION BY A -OXA PUFA Downloaded from

FIGURE 2. Effect of ␤-oxa-21:3n-3 pretreatment time and concentra- tion on calcium ionophore A23187-enhanced LTB4 production by neutro- phils. Neutrophils (5 ϫ 106) were preincubated with or without ␤-oxa-21:

3n-3 (20 ␮M) in 0.5 ml of HBSS at 37¡C for 0Ð60 min (A) or were http://www.jimmunol.org/ preincubated with ␤-oxa-21:3n-3 (0Ð40 ␮M) in 0.5 ml of HBSS at 37¡C for 15 min (B). The cells were then incubated in a 1-ml final volume of HBSS containing the calcium ionophore A23187 (5 ␮M) at 37¡C for 30 min. The amount of LTB4 in the medium was determined as described in Materials and Methods. Each point represents the mean Ϯ SEM for three determinations. These experiments were conducted three times with similar p Ͻ 0.0001 (for significant differences between ,ءء ;p Ͻ 0.001 ,ء :results. A pretreatment with and without ␤-oxa-21:3n-3 at a particular time point, by p Ͻ 0.01 ,ءء ;p Ͻ 0.05 ,ء :two-tailed Student’s t test for unpaired data). B (for significant differences between pretreatment with ␤-oxa-21:3n-3 and by guest on September 25, 2021 control, by one-way ANOVA, followed by Dunnett’s test for multiple FIGURE 3. Effect of ␤-oxa-21:3n-3 on the production of [3H]5-HETE 3 3 comparisons). The basal production of LTB4 by the cells was negligible (A) and [ H]LTB4 (B)by[H]20:4n-6-prelabeled neutrophils challenged and was not significantly affected by ␤-oxa-21:3n-3 pretreatment. with the calcium ionophore A23187. Neutrophils (2 ϫ 107, prelabeled with [3H]20:4n-6) were preincubated with or without ␤-oxa-21:3n-3 (20 ␮M) in 2 ml of HBSS at 37¡C for 15 min. The cells were then incubated in a 4-ml final volume (i.e., 5 ϫ 106/ml) of HBSS containing the calcium ionophore A23187 (5 ␮M) at 37¡C for up to 30 min. The radioactivity associated with activation during the 75 min in preincubation with [3H]20:4n-6. 5-HETE and LTB4 was determined as described in Materials and Methods. The level of 5-HETE and LTB4 remained constant during 10- to Each point represents the mean Ϯ SEM for three determinations. This p Ͻ ,ءء ;p Ͻ 0.01 ,ء .30-min incubation, instead of the expected decrease if LTB4 was experiment was performed twice with similar results ␻-oxidized. This is either due to an excess amount being produced 0.001 (for significant differences between pretreatment with and without ␤ or the fact that the LTB4 measured included the two-6-trans -oxa-21:3n-3 at a particular time point, by two-tailed Student’s t test for isozymes of LTB4, which are not as well ␻-oxidized. unpaired data). C, Effect of ␤-oxa-21:3n-3 on the production of [3H]5- 3 3 Interestingly, pretreatment with ␤-oxa-21:3n-3 suppressed the HETE and [ H]LTB4 by neutrophils challenged with [ H]20:4n-6 and the 7 production of [3H]5-HETE and [3H]LTB by leukocytes chal- calcium ionophore A23187. Neutrophils (2 ϫ 10 ) were preincubated with 4 ␤ ␮ lenged simultaneously with [3H]20:4n-6 and the calcium iono- or without -oxa-21:3n-3 (20 M) in 2 ml of HBSS at 37¡C for 15 min. phore (Fig. 3C). This suggested that the ␤-oxa-fatty acid could be The cells were then incubated in a 4-ml final volume of HBSS containing [3H]20:4n-6 (2 ␮Ci, 5 ␮M) and the calcium ionophore A23187 (5 ␮M) at directly inhibiting the 5-lipoxygenase enzymes. 37¡C for 30 min. The radioactivity associated with 5-HETE and LTB4 was determined as described in Materials and Methods. Each bar represents the mean Ϯ SEM for three determinations. This experiment was conducted Inhibition of 5-lipoxygenase activity by ␤-oxa-21:3n-3 p Ͻ 0.0001 (for significant differences ,ء .three times with similar results Fig. 4 shows that the ␤-oxa-PUFA substantially inhibited the ac- between pretreatment with and without ␤-oxa-21:3n-3, by two-tailed Stu- ϭ Ϯ ␮ Ϯ tivity of purified 5-lipoxygenase (IC50 26 2 M; mean dent’s t test for unpaired data). SEM of three analyses). Conversely, ␤-oxa-21:3n-3 had no signif- icant effect on the activity of purified LTA4 hydrolase, cyclooxy- genase-1, or cyclooxygenase-2 (results not shown). It remains to Incorporation into phospholipids and neutral lipids be determined whether ␤-oxa-21:3n-3 can inhibit the activity of A substantial amount of ␤-oxa-21:3n-3 was taken up by neutro-

LTA4 synthase in the neutrophil LTB4 pathway. Overall, the data phils during the first 15 min of incubation and thereafter plateaued show that ␤-oxa-21:3n-3 is a direct inhibitor of 5-lipoxygenase in up to 60 min. The ␤-oxa-PUFA taken up by the leukocytes after 60 human neutrophils, leading to reduced formation of 5-HETE and, min was mainly present in the unesterified form, with smaller consequently, LTB4. amounts incorporated in neutral lipids (predominantly cholesterol The Journal of Immunology 4777

Metabolism of ␤-oxa-21:3n-3 in stimulated neutrophils After neutrophils were pretreated with ␤-oxa-21:3n-3 for 15 min, followed by stimulation with A23187 for 30 min, a small amount of a single oxygenated fatty acid product was observed. The total ion chromatogram produced by electrospray MS analysis of the product showed a molecular ion at m/z 337 (Mϩ Ϫ1), which was expected for a monohydroxylated analog of ␤-oxa-21:3n-3. A daughter ion was found at m/z 238, corresponding to the loss of a

C6H11O fragment resulting from C15-C16 bond cleavage. This fragment unambiguously confirms the identification of the oxy- genated product with a monohydroxyl group at carbon atom 16. Pretreatment of the leukocytes with NDGA (lipoxygenase inhibi- tor) markedly suppressed the formation of the oxygenated product, whereas indomethacin (a cyclooxygenase inhibitor) had no effect. In the absence of calcium ionophore stimulation, a negligible ␤ FIGURE 4. Effect of -oxa-21:3n-3 on the activity of purified 5-lipoxy- amount of the product was recovered. Together these results pro- genase. The influence of ␤-oxa-21:3n-3 (0Ð100 ␮M) on purified 5-lipoxy- vide evidence that neutrophils convert ␤-oxa-21:3n-3 to a 16-mo- genase activity (using 30 ␮M 20:4n-6 as substrate) was determined as Ϯ nohydroxylated derivative (16-OH-␤-oxa-21:3n-3) by the lipoxy-

described in Materials and Methods. Each point represents the mean Downloaded from ␤ p Ͻ 0.05; genase enzyme pathway. Derivatization of -oxa-21:3n-3 to ,ء .SEM for three separate experiments performed in triplicate p Ͻ 0.01 (for significant differences between pretreatment with ␤-oxa- methylated, saturated, 16-monohydroperoxy or 16-monohydroxy ,ءء 21:3n-3 and controls, by one-way ANOVA, followed by Dunnett’s test for forms resulted in the loss of its effect (Fig. 5), indicating that the multiple comparisons). structure of the parent molecule is mandatory for the inhibitory effect.

Discussion http://www.jimmunol.org/ esters and diacylglycerol) and phospholipids (predominantly phos- Data from investigations examining the effects of addition of dif- phatidylinositol, phosphatidylcholine, and phosphatidylethano- ferent types of fatty acids to neutrophils on LTB production dem- lamine; Table I). The ␤-oxa-21:3n-3 was found to be exclusively 4 onstrated that 20:4n-6 significantly increased production compared localized in the sn-2 position of neutrophil phosphatidylinositol, with the n-3 fatty acids, 20:5n-3 and 22:6n-3, which significantly phosphatidylcholine, and phosphatidylethanolamine. The neutro- decreased production. This is consistent with reports in the liter- phils did not modify ␤-oxa-21:3n-3 by chain elongation, desatu- ature that the lipoxygenase form different and less proinflamma- ration, or ␤-oxidation (data not shown). We previously demon- tory metabolites from these n-3 fatty acids (44). Presumably this strated that neutrophils can elongate the natural PUFA, 20:4n-6, and tetracosatetraenoic acid (24:4n-6), by up to four carbon units, by guest on September 25, 2021 but have a low capacity to ␤-oxidize and desaturate these fatty acid substrates (36). After ␤-oxa-21:3n-3 was administered to mice and rats by ga- vage or ip injection (daily doses of 100 mg/kg body weight for 3 days), it was found to be incorporated into the lipids of various tissues, including liver, lungs, kidneys, heart, spleen, brain, adi- pose tissue, and blood (data not presented). The animals given ␤-oxa-21:3n-3 maintained normal body weight and retained nor- mal liver and kidney function (29).

Table I. Incorporation of ␤-oxa-21:3n-3 into phospholipids and neutral lipids of neutrophilsa

Lipid Recovered ␤-Oxa-21:3n-3 (% of total) FIGURE 5. Effect of ␤-oxa-21:3n-3 derivatives on LTB production by Phosphatidylcholine 6.6 Ϯ 0.3 4 Ϯ neutrophils stimulated with the calcium ionophore A23187. Neutrophils Phosphatidylethanolamine 6.0 0.3 ϫ 6 ␤ ␮ ␤ Phosphatidylinositol 10.5 Ϯ 0.6 (5 10 ) were pretreated with -oxa-21:3n-3 (20 M), -oxa-21:3n-3 ␮ Phosphatidylserine 5.0 Ϯ 0.8 derivatives (20 M), or diluent (control) in 0.5 ml of HBSS at 37¡C for 15 Phosphatidic acid 1.4 Ϯ 0.3 min. The cells were then incubated in a 1-ml final volume of HBSS con- Sphingomyelin 1.7 Ϯ 0.5 taining the calcium ionophore A23187 (5 ␮M) at 37¡C for 30 min. The Ϯ Monoacylglycerol 3.2 0.6 mass of LTB4 in the medium was determined as described in Materials and Diacylglycerol 12.3 Ϯ 1.3 Methods. Each bar represents the mean Ϯ SEM for three determinations. p Ͻ ,ء .Triacylglycerol 2.1 Ϯ 0.5 This experiment was performed three times with similar results Ϯ Cholesterol ester 13.3 1.8 0.01 (for significant differences between pretreatment with compounds and Free fatty acid 37.9 Ϯ 0.2 controls, by one-way ANOVA, followed by Dunnett’s test for multiple a ϫ 7 ␤ ␮ Neutrophils (4 10 ) were incubated with -oxa-21:3n-3 (20 M) or diluent comparisons). The basal production of LTB4 by the cells was negligible (control) in 4 ml of HBSS at 37¡C for 60 min. The amount of ␤-oxa-21:3n-3 asso- and was not significantly affected by agonist pretreatment. ␤-oxa-21:3n- ciated with phospholipids and neutral lipids of the cells was determined as described ␤ ␤ ␤ Ϯ 3ME, -oxa-21:3n-3 methyl ester; -oxa-21:0, saturated -oxa-21:3n-3; in Materials and Methods. The results represent the mean SEM of three separate ␤ ␤ ␤ experiments performed in triplicate and are expressed as a percentage of the total -oxa-21:3n-3OOH, 16-monohydroperoxy- -oxa-21:3n-3; -oxa-21:3n- recovered ␤-oxa-21:3n-3. 3OH, 16-monohydroxy-␤-oxa-21:3n-3. ␤ 4778 INHIBITION OF NEUTROPHIL LTB4 PRODUCTION BY A -OXA PUFA explains many of the beneficial effects seen with n-3 fatty acids, highly likely that the observation that the ␤-oxa 21:3n-3 inhibition including fish oil given to animals or humans experiencing inflam- of LTB4 production is responsible for the previously observed ef- matory diseases (40). Interestingly our novel ␤-oxa 21:3n-3 was fects of this fatty acid on acute inflammation induced by carra- much more potent than both of the above n-3 in inhibiting geenan (29), in which neutrophils play a dominant role (45, 46). ␤ LTB4 production. While 22:6n-3 and 20:5n-3 inhibited production Our data together with our previous findings that -oxa-21:3n-3 by 25%, ␤-oxa 21:3n-3 caused Ͼ95% inhibition. Thus, this ␤-oxa- inhibited carrageenan-induced paw inflammation (29) suggest an fatty acid was as efficacious as Zileuton and NDGA in inhibiting important role for the 5-lipoxygenase pathway in this inflamma- ␤ LTB4 production. The differences in activity between 22:6n-3 and tory reaction (47). -Oxa-21:3n-3 was also shown (29) to inhibit the novel n-3 fatty acid also correlate with our previous findings the delayed-type hypersensitivity response and in vitro inhibition that the latter is a stronger anti-inflammatory agent than the former of T lymphocyte function. However, this appeared to be due to a (29). It is therefore tempting to speculate that the ␤-oxa fatty acid different mechanism, involving inhibition of protein kinase C and is likely to be more useful than the fish oil fatty acids for treating the mitogen-activated protein kinase, extracellular signal-regulated inflammatory diseases. kinase 1/2, particularly as evidence suggests that T lymphocytes The role of the structure of ␤-oxa 21:3n-3 in the inhibition of lack lipoxygenase activity (29). ␤ ␤ LTB4 production was evaluated, and it was evident that the un- Our observations suggest that -oxa-21:3n-3 and other -oxa- saturation was important, since a ␤-oxa 21:0 fatty acid was not PUFA are potent, direct inhibitors of 5-lipoxygenase activity in active. However, the position of the double bonds made only a human leukocytes, resulting in reduced production of 5-HETE and ␤ ␤ slight difference in activity, where -oxa 21:3n-6 was almost as LTB4. Thus, these -oxa-PUFA will provide new avenues to de- active as the n-3 equivalent. In contrast, methylation of the car- velop improved therapeutic strategies for inflammatory diseases, Downloaded from boxyl group, hydroxylation, and formation of the hydroperoxy all such as asthma, rheumatoid arthritis, systemic lupus erythematosis, resulted in the total loss of activity of the molecule. This demon- and atherosclerosis. The feasibility of using this fatty acid to treat strates that the unoxidised parent ␤-oxa-21:3n-3 is the inhibitory these diseases is supported by our findings of the lack of toxicity agent, which is consistent with our finding that most of the added (29) and ability to incorporate into lipids of tissues following oral fatty acid was found as a free fatty acid in stimulated neutrophils. administration.

The inhibition of production of LTB4 was upstream of LTB4, http://www.jimmunol.org/ since production of 5-HETE was similarly inhibited by ␤-oxa 21: Acknowledgments 3n-3. Since this fatty acid also dramatically inhibited the oxidation We thank Gina Parashakis for her excellent technical assistance, and Prof. of radiolabeled 20:4n-6 to the above metabolites in neutrophils Alf Poulos for invaluable advice during the course of this work. when both the labeled fatty acid and calcium ionophore were added together, it implies a direct effect on the lipoxygenase en- References zymes. Using the purified enzyme, this fatty acid was shown to 1. Van Golde, L. M. G., and S. G. Van den Bergh. 1977. General pathways in the directly inhibit the 5-lipoxygenase enzyme activity. Indeed, it metabolism of lipids in mammalian tissues. In Lipid Metabolism in Mammals, Vol. 1. F. Snyder, ed. Plenum Press, New York, p. 1. showed selectivity for this enzyme, since it did not inhibit purified 2. Weller, P. 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