Human neutrophils convert the sebum-derived polyunsaturated fatty acid Sebaleic acid to a potent granulocyte chemoattractant

Abstract

Sebaleic acid (5,8-octadecadienoic acid) is the major polyunsaturated fatty acid in human sebum and skin surface lipids. The objective of the present study was to investigate the metabolism of this fatty acid by human neutrophils and to determine whether its metabolites are biologically active. Neutrophils converted sebaleic acid to four major products, which were identified by their chromatographic properties, UV absorbance, and mass spectra as 5-hydroxy-(6E,8Z)-octadecadienoic acid (5-HODE), 5-oxo-(6E,8Z)-octadecadienoic acid (5-oxo-ODE), 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid, and 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid. The identities of these metabolites were confirmed by comparison of their properties with those of authentic chemically synthesized standards. Both neutrophils and human keratinocytes converted 5-HODE to 5-oxo-ODE. This reaction was stimulated in neutrophils by phorbol myristate acetate and in keratinocytes by oxidative stress (t-butyl-hydroperoxide). Both treatments dramatically elevated intracellular levels of NADP(+), the cofactor required by 5-hydroxyeicosanoid dehydrogenase. In keratinocytes, this was accompanied by a rapid increase in intracellular GSSG levels, consistent with the involvement of glutathione peroxidase. 5-Oxo-ODE stimulated calcium mobilization in human neutrophils and induced desensitization to 5-oxo-6,8,11,14-eicosatetraenoic acid but not leukotriene B(4), indicating that this effect was mediated by the OXE receptor. 5-Oxo-ODE and its 8-trans isomer were equipotent with 5-oxo-6,8,11,14-eicosatetraenoic acid in stimulating actin polymerization and chemotaxis in human neutrophils, whereas 5-HODE, 5-oxo-18-hydroxy-(6E,8Z)-octadecadienoic acid, and 5S,18-dihydroxy-(6E,8Z)-octadecadienoic acid were much less active. We conclude that neutrophil 5-lipoxygenase converts sebaleic acid to 5-HODE, which can be further metabolized to 5-oxo-ODE by 5-hydroxyeicosanoid dehydrogenase in neutrophils and keratinocytes. Because of its chemoattractant properties, sebum-derived 5-oxo-ODE could be involved in neutrophil infiltration in inflammatory skin diseases.

FIGURE 1.

Chemical synthesis of sebaleic acid and its metabolites. Sebaleic acid (A), 5-HODE (B), the ω-oxidation products 5,18-diHODE and 5-oxo-18-HODE (C), 5-oxo-ODE (D), and 8-trans-5-oxo-ODE (E) were prepared by total chemical synthesis as shown. Full details of the conditions used for these syntheses will be published elsewhere.

FIGURE 2.

Chromatographic profile of sebaleic acid metabolites formed by neutrophils. A, human neutrophils (5 × 10⁶ cells in 1 ml) were preincubated for 5 min with PMA (50 nm) and then incubated with sebaleic acid (50 μm) and A23187 (5 μm) for a further 40 min at 37 °C. The products were analyzed by automated precolumn extraction/RP-HPLC with 13-HODE (100 ng) as an internal standard (int std). The stationary phase was a Waters Nova-Pak C18 column (3.9 × 150 mm), whereas the mobile phase was a linear gradient over 40 min between water/acetonitrile/methanol/acetic acid (60:30:10:0.02) and water/acetonitrile/methanol/acetic acid (10:38:52:0.02) with a flow rate of 1 ml/min. The top chromatogram shows the profile for UV absorbance at 235 nm, whereas the bottom chromatogram shows absorbance at 280 nm. 5oETE, 5-oxo-ETE; 20h-LTB₄, 20-hydroxy-LTB₄. B, UV spectra of metabolites I (– – –), II (——), III (–··–), and IV (–·–).

FIGURE 3.

Product ion mass spectra of sebaleic acid metabolites. Product ion spectra were obtained following collision-induced dissociation of the M-1 ion from neutrophil-derived metabolite I (5-HODE) (A) and metabolite II (5-oxo-ODE) (B), chemically synthesized 5-HODE (C) and 5-oxo-ODE (D), and neutrophil-derived metabolite III (5,18-diHODE) (E) and metabolite IV (5-oxo-18-HODE) (F). All of the above compounds were prepared as described under “Experimental Procedures” and analyzed by FTMS.

FIGURE 4.

Evidence for the formation of 5-HODE by 5-LO. A mixture of the methyl esters of chemically synthesized 5R-HODE and 5S-HODE (A), the methyl ester of chemically synthesized 5R-HODE (B), and 5-HODE methyl ester, isolated following incubation of neutrophils with sebaleic acid and A23187 as described under “Experimental Procedures” (C) were analyzed by HPLC on a column of Chiralpak AD. The brackets in each chromatogram show the retention times of 5R-HODE and 5S-HODE. D and E show the effects of different concentrations of MK-886 and zileuton, respectively, on the formation of 5-HODE (▴) and 5-oxo-ODE (○) by neutrophils incubated with sebaleic acid in the presence of A23187 and PMA as described under “Experimental Procedures.” The values are means ± S.E. of data from four independent experiments with different donors.

FIGURE 5.

Formation of 5-HODE metabolites and NADP⁺ by PMA-stimulated neutrophils. A, high performance liquid chromatogram of the products obtained after preincubation of neutrophils (2 × 10⁶ cells in 1 ml) with PMA (50 nm) for 5 min, followed by incubation with 5-HODE (4 μm) for 20 min. The products were analyzed by precolumn extraction/RP-HPLC using a Waters Nova-Pak C18 column with a mobile phase consisting of a linear gradient between water/acetonitrile/acetic acid (65:35:0.02) and water/acetonitrile/acetic acid (25:75:0.02) at a flow rate of 1 ml/min. B and C, time courses for the formation of 5-oxo-ODE (5oODE)(•), 5-oxo-18-HODE (5o-18h; ○), and 5,18-diHODE (5,18-dh; ▵) by neutrophils preincubated for 5 min with either vehicle (B) or 50 nm PMA (C), followed by incubation with 5-HODE (4 μm) for various times. The products were analyzed by RP-HPLC as described above. The results are means ± S.E. (n = 4). D, time course for the effect of PMA (50 nm; added at 0 min) on intracellular NADP⁺ levels in neutrophils. NADP⁺ was measured as described under “Experimental Procedures.” Results are means ± S.E. (n = 5). int std, internal standard.

FIGURE 6.

Effects of tBuOOH on 5-oxo-ODE synthesis and GSSG and NADP⁺ levels in human keratinocytes. A, keratinocytes (3 × 10⁵ cells/well) were incubated with 5-HODE (100 μm) and either vehicle (○) or tBuOOH (•) for various times at 37 °C. 5-Oxo-ODE was measured by RP-HPLC as described in the legend to Fig. 5. The results are means ± S.E. (n = 5). B, effects of tBuOOH, added at 0 min, on the intracellular levels of GSSG (•) and NADP⁺ (▵), measured as described under “Experimental Procedures,” in keratinocytes. The values are means ± S.E. (n = 4).

FIGURE 7.

Effects of 5-oxo-ODE on intracellular calcium levels in neutrophils. Indo-1-loaded neutrophils were treated with either 5-oxo-ODE (5oODE) (10 nm)(A) or vehicle (B), followed by 5-oxo-ETE (5oETE) (10 nm), LTB₄ (10 nm), and ionomycin (1 μm). Fluorescence was measured as described under “Experimental Procedures.” The results are representative of three similar experiments.

FIGURE 8.

Effects of sebaleic acid metabolites on actin polymerization in neutrophils. Neutrophils were treated for 20 s with various concentrations of 5-oxo-ODE (5oODE)(•), 8-trans-5-oxo-ODE (8t-5oODE)(▿), 5-oxo-ETE (5oETE) (□), 5-oxo-18-HODE (5o-18h)(○), 5-HODE (▴), and 5,18-diHODE (5,18-dh)(▵), and actin levels were measured by flow cytometry as described under “Experimental Procedures.” All values are means ± S.E. (n = 6).

FIGURE 9.

Chemoattractant effects of 5-oxo-ODE. Chemotactic responses of neutrophils to 5-oxo-ODE (•), 8-trans-5-oxo-ODE (▿), and 5-oxo-ETE (□) were measured using microchemotaxis chambers as described under “Experimental Procedures.” 5oODE, 5-oxo-ODE; 8t-5oODE, 8-trans-5-oxo-ODE; 5oETE, 5-oxo-ETE. All values are means ± S.E. (n = 5) and are expressed as percentages of the maximal response to 5-oxo-ETE, which was 230 ± 32 cells/high power field, compared with 11 ± 2 cells/high power field for vehicle-treated controls.

FIGURE 10.

Metabolism of sebaleic acid by human neutrophils. Both 5-HODE and 5-oxo-ODE are assumed to be converted to 18-hydroxy products by the action of LTB₄ 20-hydroxylase (CYP4F3). Although 8-trans-5-oxo-ODE was not identified as a product of sebaleic acid metabolism by neutrophils, this substance has been reported to be a constituent of maca tubers.