Published online before print July 8, 2005
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Divisions of Infectious Diseases and
* Pulmonary and Critical Care Medicine, Department of Internal Medicine, Medical School, University of Michigan, Ann Arbor; and
Department of Immunology, Institute of Biomedical Sciences IV, University of São Paulo, Brazil
1Correspondence: Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Health System, 6301 MSRB III, Box 0642, 1150 W. Medical Center Drive, Ann Arbor, MI 48109-0642. E-mail: petersm{at}umich.edu
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40% when compared with untreated controls. LTB4 was the moiety responsible for H2O2 production, as revealed by studies using receptor antagonists and its exogenous addition. LTB4 itself also promoted p47phox phosphorylation and translocation. These results identify a heretofore unrecognized role for activation of 5-LO and subsequent production of LTB4 in stimulation of PMN NADPH oxidase activation by PUFAs.
Key Words: PMN • PUFAs • lipid mediators • NADPH oxidase • BLT1
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) end into those of the n-3 (-3) and those of the n-6 (-6) series. PUFAs are essential constituents of mammalian diets, required for maintenance of normal cell structure and function. Examples of n-6 PUFAs are linoleic acid (LA; 18:2n-6) and arachidonic acid (AA; 20:4n-6). AA and LA are liberated from membrane phospholipids by the action of phospholipase A2 (PLA2) enzymes. AA serves the well-characterized role of substrate for the generation of bioactive eicosanoids, including prostaglandins and leukotrienes (LTs). Besides its role as a precursor for eicosanoids, endogenous AA may function as an intracellular signaling molecule [2
, 3
] or as a cell activator when released into the extracellular space.
Numerous signaling components have been reported to be activated by exogenous AA. These include guanosine 5'-triphosphate-binding proteins [4
], plasma membrane Ca++ channels [5
], extracellular signal-regulated kinase 1/2 (ERK 1/2) [6
], Raf-1/mitogen-activated protein kinase kinase (MEK) [7
], c-Jun N-terminal kinase [8
], phosphatidylinositol 3-kinase [9
], PLC [9
], nuclear factor-
B [10
], and protein kinase C (PKC) [11
]. In neutrophils [polymorphonuclear leukocytes (PMNs)], it has been suggested that activation of ERK 1/2 and Raf-1/MEK by AA could be mediated by formation of its lipoxygenase (LO) metabolic products [6
, 7
]. One of the most well-studied actions of AA is the activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in cell-free systems as well as intact PMNs [4
, 5
, 12
13
14
15
16
17
18
19
]. The NADPH oxidase complex is formed by the cytosolic proteins p47phox, p40phox, p67phox, and Rac-1 or -2 and the membrane-associated components gp91phox, p22phox, and Rap-1 [20
]. Upon cell activation, the cytosolic components are phosphorylated by specific serine kinases. This leads to structural modification of the phox proteins, allowing their translocation to the cell membrane [20
]. Most activators of NADPH oxidase, such as cytokines, lipids, and bacteria, act via receptor-mediated signaling pathways; however, lipids, such as AA, LA, diacylglycerol, or phosphatidic acid, may also interact directly with p47phox or p67phox proteins by unmasking their membrane-binding domains [19
, 21
] or may interact with other relevant regulatory components [22
23
24
25
26
27
].
AA is converted to LTs by 5-LO, and the major products formed in PMNs are LTB4 and 5-hydroxy-eicosatetraynoic acid (5-HETE) [28
]. It is important that LTB4 has been shown to activate NADPH oxidase [29
, 30
], although the mechanism of this activation remains unknown. However, we recently demonstrated that in rat alveolar macrophages, LTB4 activates NADPH oxidase through phosphorylation and translocation of p47phox to the membrane, a process that was itself dependent on PKC-
activity [31
]. It is unclear whether the ability of AA to activate NADPH oxidase depends, in part, on its metabolism to LTB4. Conversely, as LTB4 activates cytosolic PLA2 (cPLA2) [32
], it is possible that NADPH oxidase activation depends on LTB4-stimulated AA. As AA and LTB4 can stimulate Ca++ release [33
, 34
] as well as PLC and PKC activities [9
, 11
, 30
, 35
], these actions may affect NADPH oxidase indirectly as well. Thus, the goals of this work were to evaluate the role of 5-LO metabolites in PUFA-induced NADPH oxidase activation in PMNs and to determine the molecular mechanisms involved in these actions. Herein, we report that LTB4 is a significant mediator of the ability of AA to activate NADPH oxidase in PMNs. LTB4 participates in NADPH oxidase activation by modulating p47phox phosphorylation and translocation to the membrane. To our knowledge, this is the first report showing that LTB4 is able to directly affect the assembly of NADPH oxidase components in PMNs.
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Animals
5-LO knockout (KO; 129-Alox5tm1Fun) [36
] and strain-matched wild-type (WT) sv129 mice were bred in the University of Michigan Unit for Laboratory Animal Medicine (Ann Arbor) from breeders obtained from Jackson Laboratories (Bar Harbor, ME), and female Wistar rats were obtained from Charles River Laboratories (Portage, MI). The University Committee on Use and Care of Animals approved animal protocols.
Cell harvest
Glycogen-elicited rat or murine PMNs were obtained following injection of 20 mL 4% glycogen (Sigma Chemical Co.) into the peritoneal cavity. After 5–6 h, the peritoneal exudates were harvested by lavage with phosphate-buffered saline (PBS). Contaminating red blood cells were lysed with H2O, and the cells were washed 2x with PBS. The percentage of PMNs was determined microscopically using a modified Wright-Giemsa stain, and a typical experiment yielded 90% PMNs.
Cell viability
No experimental compounds or vehicle showed any adverse effects on PMN viability as determined by a cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (data not shown).
Hydrogen peroxide (H2O2) detection
Rat PMNs (5x105/well) were plated in 96-well dishes as described above. H2O2 production was assessed in a HRP-coupled reaction using Amplex Red as a probe, according to the manufacturer (Molecular Probes). To assess the role of 5-LO metabolites in H2O2 production stimulated by PUFAs, the cells were first pretreated with zileuton, AA-861, MK886, MK571, or CP105,696 for 20 min followed by the addition of the Amplex Red solution containing AA or LA. The solution containing 50 µM Amplex Red reagents and 10 U/mL HRP was prepared in PBS, 0.1 mL was added to PMNs at 37°C for 30 min, and the H2O2 levels were determined colorimetrically (absorbance at 560 nm).
LTB4 measurement
Rat PMNs (5x105 cells/well) were cultured in 96-well plates in DMEM. Cultures were then incubated for 5, 15, and 30 min with 20 µM AA to stimulate LT production. Culture supernatants were collected, and LTB4 levels were quantified by enzyme immunoassay, according to the manufacturer (Assay Designs, Ann Arbor, MI).
Fractionation, immunoprecipitation, and Western blotting
PMNs (1x107) were plated in six-well tissue-culture dishes and pretreated with AA-861 or CP105,696 for 20 min, followed by stimulation for 5 min with 20 µM AA or vehicle control. After this, PMNs were lysed by sonication in ice-cold lysis buffer containing 150 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM sodium orthovanodate, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, and 1 µg/mL leupeptin, followed by ultracentrifugation at 100,000 g for 20 min at 4°C. The cytosolic (soluble) fraction was harvested, and the membrane (insoluble) fraction was washed and subjected to another ultracentrifugation step as described above. The resulting pellet was resuspended in lysis buffer and sonicated. Protein concentrations were determined by a modified Coomassie dye-binding assay (Pierce Chemical Co., Rockford, IL). The cytosolic fraction was used for immunoprecipitation as described previously [37
] with some modifications. The fraction was incubated overnight at 4°C with anti-p47phox antibody (1:80; Upstate Biotechnology, Lake Placid, NY). Protein A-Sepharose was added to each sample and incubated for 3 h with rotation at 4°C. The beads were washed briefly 3x with lysis buffer without Triton X-100, and samples containing 20 µg protein were separated on 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred to nitrocellulose membranes. Following blocking with 5% nonfat milk, membranes were probed with antibodies directed against p47phox (1:500 dilution; Upstate Biotechnology) or antiphosphoserine (1:900; KI-191, clone 1C8; Biomol) for 90 min, followed by peroxidase-conjugated goat anti-rabbit (Amersham, Piscataway, NJ) or anti-mouse secondary (1:5000; Zymed, South San Francisco, CA) and developed using enhanced chemiluminescence detection (Amersham).
Statistical analysis
Data are represented as mean ± SE and were analyzed with the Prism 3.0 statistical program (GraphPad Software, San Diego, CA). Comparisons between two experimental groups were performed with Student’s t-test. Comparisons among more than or equal to three experimental groups were performed with ANOVA followed by the Bonferroni analysis. Differences were considered significant if P 
0.05. All experiments were performed on more than or equal to three separate occasions unless otherwise specified.
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Figure 1. PUFAs stimulate PMN H2O2 release in a dose-dependent manner. H2O2 from glycogen-elicited rat PMNs was measured 30 min after stimulation with AA (A) or LA (B). H2O2 concentrations were determined using Amplex Red as a probe as described in Materials and Methods. Data represent mean ± SE from quadruplicate values from one experiment representative of a total of four. *, P< 0.05,versus control by ANOVA.
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Figure 2. PMN H2O2 production in response to PUFAs is dependent on NADPH oxidase activity. PMNs were treated with the NADPH oxidase inhibitor DPI (10 µM) for 20 min and then stimulated for 30 min with 20 µM AA or 20 µM LA. H2O2 production was determined using Amplex Red as a probe, as described in Materials and Methods. Data represent mean ± SE from quadruplicate values from one experiment representative of a total of four. *, P < 0.05, versus control and #, P< 0.05 versus PUFA alone by ANOVA.
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Our first approach was to determine whether endogenous 5-LO metabolites play a role in PUFA-stimulated H2O2 production. Rat PMNs were pretreated with either of two inhibitors of LT synthesis, namely, the 5-LO inhibitor zileuton (10 µM) or the FLAP inhibitor MK886 (1 µM), for 20 min before activation with AA or LA (20 µM). As observed in Figure 3A
and 3C
, zileuton and MK886 treatment decreased by 40% the AA- or LA-induced H2O2 production as compared with untreated cells. Similar experiments using cells from 5-LO-deficient mice confirmed these results (Fig. 3B
and 3D)
. It is interesting that when the PMNs were stimulated with a lower dose of AA (5 µM; a concentration of AA that is perhaps more relevant to its possible function as an endogenous second messenger), the role of endogenous 5-LO metabolites in inducing H2O2 generation was even more pronounced. For example, stimulation of H2O2 generation with 20 µM AA was inhibited by 40% by the 5-LO inhibitor AA-861, but with 5 µM AA, the inhibition was 75% when compared with untreated control (control, 2.93±0.129; AA, 11.23±0.134; AA+AA-861, 5.080±0.240 nM H2O2/2x105 PMN/30 min; P<0.05 for AA+AA-861 vs. AA alone). Taken together, these data indicate that H2O2 production stimulated by PUFAs in glycogen-elicited PMNs is dependent, in part, on endogenous 5-LO products.
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Figure 3. H2O2 production in response to PUFAs in LT-deficient PMNs. Glycogen-elicited rat PMNs were pretreated with the 5-LO inhibitor zileuton (10 µM) or the FLAP inhibitor MK886 (1 µM) for 20 min and then stimulated for 30 min with 20 µM AA (A) or 20 µM LA (C). Glycogen-elicited PMNs from WT or 5-LO KO mice were stimulated with AA (B) or LA (D) for 30 min. H2O2 concentrations were determined using Amplex Red as a probe as described in Materials and Methods. Data represent the mean ± SE of quadruplicate values from one experiment representative of a total of four. *, P< 0.05 versus control and #, P< 0.05 versus PUFA alone by ANOVA.
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Figure 4. Role of LT receptors in PMN H2O2 production by PUFAs. Glycogen-elicited rat PMNs were treated with the BLT1 antagonist CP105,696 (10 µM) or the cysLT1 antagonist MK571 (10 µM) and then stimulated for 30 min with 20 µM AA (A) or 20 µM LA (B). H2O2 production was determined using Amplex Red as a probe as described in Materials and Methods. Data represent mean ± SE of quadruplicate values from one experiment representative of a total of four. *, P< 0.05 versus control and # P< 0.05, versus PUFA alone by ANOVA.
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Figure 5. AA stimulates LTB4 production followed by H2O2 secretion in PMN from rats. (A) H2O2 levels were measured as described in Materials and Methods after stimulation with different LTB4 concentrations. (B) LTB4 and H2O2 levels were measured as described in Materials and Methods in PMN supernatants at different time-points following addition of 20 µM AA. Data represent mean ± SE of quadruplicate values from one experiment representative of a total of four. *, P< 0.05 versus control by ANOVA.
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Next, we wished to investigate whether it was possible to restore H2O2 secretion with exogenous LTB4 in PMNs treated with inhibitors of endogenous LT synthesis and if the LTB4 effect was a result of interaction with the BLT1 receptor. We therefore pretreated PMNs for 20 min with the 5-LO inhibitor AA-861 (10 µM; Fig. 6A ) or the BLT1 antagonist CP105,696 (10 µM; Fig. 6B ), followed by stimulation with AA and/or LTB4. Our results show that exogenous LTB4 restored the inhibition of H2O2 generation in PMNs treated with the 5-LO inhibitor. In contrast, the addition of LTB4 to AA-treated PMNs incubated with the BLT1 antagonist did not overcome this inhibition. These results indicate that endogenous LTB4 is important in NADPH oxidase activation by AA, and this effect is mediated by interaction with the BLT1 receptor.
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Figure 6. Role of BLT1 receptor in mediating H2O2 stimulation. Glycogen-elicited rat PMNs were treated with the 5-LO inhibitor AA-861 (10 µM; A) or the BLT1 antagonist CP105,696 (10 µM; B) and then stimulated for 30 min with 20 µM AA and/or 10 nM LTB4. H2O2 production was determined using Amplex Red as a probe as described in Materials and Methods. Data represent mean ± SE of quadruplicate values from one experiment representative of a total of four. *, P< 0.05 versus control; #, P< 0.05 versus AA; and &, P< 0.05 versus LTB4 by ANOVA.
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Figure 7. AA induces phosphorylation and translocation of p47phox in glycogen-elicited rat PMNs in a 5-LO- and BLT1-dependent manner. PMNs (4x106/well) were pretreated or not with CP105,696 or AA-861, stimulated by 20 µM AA or 10 nM LTB4 for 5 min, and then harvested and fractionated as described in Materials and Methods. The cytosolic fraction was immunoprecipited, and immunoprecipited protein was immunoblotted using antiphosphoserine (1:900), and phosphorylated protein is designated p-p47phox (A). The total cytosolic fraction (B) and membrane fraction (C) were probed for total (T) p47phox (1:500). Experiment is representative of a total of two.
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PUFAs are involved in inflammatory conditions, such as modulation of vascular contraction, chemotaxis, cell adhesion and diapedesis, cell activation, and cell death [44 ]. AA is a pleiotropic lipid involved in inflammatory and physiological situations. A role for AA in inflammation has been suggested by studies showing that AA increases chemotaxis, granule release, and effector functions such as phagocytosis, microbicidal activity, and release of ROI in PMNs [45 ]. The role of AA as a second messenger [2 ] and its ability to activate NADPH oxidase are well-known [4 , 5 , 12 13 14 15 16 17 18 19 ]. In addition, AA is the precursor of bioactive lipids including prostanoids and LTs, which are themselves able to induce cell activation. Upon activation, the major AA metabolites produced by PMNs are LTB4 and 5-hydroxy-5-HETE [32 ], but the respective roles of AA and its metabolic products in cell signaling are incompletely understood. Thus, we sought to evaluate the role of these 5-LO products in AA and LA stimulation of NADPH oxidase activation.
We first asked if PUFAs activate NADPH oxidase by themselves or via formation of metabolic products. As expected [18
, 19
], both PUFAs were able to induce H2O2 release dose-dependently to a similar extent at concentrations similar to those observed previously. To address the role of 5-LO metabolites in AA- or LA-stimulated PMNs, we inhibited 5-LO or FLAP (both essential for LT synthesis [46
]), and we observed an inhibition in H2O2 production of 40% when compared with the untreated control. This effect was confirmed using cells obtained from genetically 5-LO-deficient mice. Although ROI can be generated by a variety of enzymatic sources [47
], the ability of the NADPH oxidase-like flavoprotein inhibitor DPI to completely attenuate ROI production in AA- or LA-stimulated PMNs suggests that PUFA effects occurred exclusively through activation of NADPH oxidase.
Our results strongly suggest that AA and LA act to increase 5-LO activity, which results in NADPH oxidase activation. These results are consistent with studies in AA-stimulated guinea pig eosinophils pretreated with the AA congener eicosatetraynoic acid (ETYA), which prevents the further metabolism of AA [48 ]. By contrast, Pompeia et al. [38 ] demonstrated that O2– production by HL-60 cells stimulated with AA was independent of 5-LO metabolites. Regarding the dependence on AA metabolites observed with exogenous LA stimulation, Alzoghaibi et al. [49 ] showed that LA-induced interleukin-8 production in human intestinal smooth muscle cells was dependent on 5-LO and cyclooxygenase metabolites. To our knowledge, however, this is the first report showing that LA induction of NADPH oxidase activity depends on 5-LO metabolites. That endogenous 5-LO activity is important for activating NADPH oxidase has been demonstrated in PMNs stimulated by myriad agonists. Maridonneau-Parini et al. [50 ] showed that opsonized zymosan, but not PMA, induced O2– generation in a manner dependent on 5-LO and cyclooxygenase products in human PMNs. Furthermore, it was observed that 5-LO metabolites are necessary for NADPH oxidase activation in PMNs stimulated by platelet-activating factor, formyl-Met-Leu-Phe, PMA, and A23187 [51 ]. Although the role of 5-LO metabolites in NADPH oxidase activation is recognized, the importance of individual LTs in PUFA-induced NADPH oxidase activation is not. When we pretreated PMNs with BLT1 or cysLT1 receptor antagonists followed by stimulation with AA or LA, we observed that only the BLT1 antagonist abolished the H2O2 secretion in PUFA-stimulated PMNs. Likewise, only LTB4, but not cysLTs, was capable of stimulating secretion of H2O2 in PMNs. To confirm that the effects of PUFAs were dependent on LTB4 interaction with BLT1, we performed "add back" experiments. The first approach was to pretreat cells with AA-861 and then add AA with or without LTB4. Our experiments clearly show that in 5-LO-inhibited cells, the addition of LTB4 restored the PMN ability to secrete H2O2. We confirmed that the LTB4 effects are mediated by interaction with BLT1, as LTB4 addition was ineffective in BLT1 antagonist-treated cells. At lower (i.e., 5 µM) AA concentrations more relevant to those produced endogenously, which might function as second messengers, we observed an even more prominent dependence on 5-LO products and BLT1 in AA-stimulated PMNs. This could reflect the ability of lower doses of AA to activate classical PKC [11 ] or Ca++ release [9 ], both of which are known to be important in the activation of 5-LO [46 ].
Results with 5-LO inhibitors and 5-LO KO cells as well as a BLT1 antagonist implicate 5-LO metabolism/BLT1 signaling in NADPH oxidase activation by AA and LA. However, we could only measure immunoreactive LTB4 production in PMNs stimulated with AA and not with LA. These results are consistent with an alternative LT derivative of LA other than LTB4 interacting with BLT1 to affect NADPH oxidase, but we are aware of no precedent regarding such a possibility. It will be of interest to clarify this in future studies.
That PMNs respond predominantly to LTB4, but not cysLTs, is in agreement with previous findings from our laboratory and others. Mancuso et al. [52 ] showed that LTB4, but not cysLTs, increased phagocytosis of opsonized Klebsiella pneumoniae in human PMNs. In addition, Palmblad et al. [53 ] showed that only LTB4 induced NADPH oxidase activation in eosinophils and PMNs. However, Larfars et al. [42 ] demonstrated that LTB4 and cysLTs were able to induce nitric oxide production in human PMNs. In our experimental system, a role for 5-LO products in PUFA stimulation of NADPH oxidase was confined to LTB4 and its interaction with BLT1.
Although AA promotes p47phox translocation in human monocytes, its ability to enhance phosphorylation of this molecule has been the subject of conflicting reports [19 ]. In eosinophils, LTB4 stimulated H2O2 production independent of AA release [29 ], and this effect was dependent on PKC, PLC, and Ca++ [30 ]. The molecular mechanism by which LTB4 activates NADPH oxidase activation in PMNs is unclear. Thus, we sought to determine the effects of LTB4 on p47phox phosphorylation and translocation in AA-stimulated PMNs. Our results show that inhibition of 5-LO metabolism or antagonism of BLT1 decreased phosphorylation and translocation of p47phox in AA-stimulated cells and that exogenous LTB4 was able to increase its phosphorylation and translocation. We have observed similar effects of LA. Together, our results suggest that LTB4 contributes to activation of the p47phox cytosolic component of NADPH oxidase in PUFA-stimulated PMNs.
Activation of p47phox is regulated by the action of a variety of kinases, including PKC [43
]. PUFAs act as second messengers, in part, by stimulating these kinases [2
]. Our present data are the first to implicate 5-LO products in PUFA-induced p47phox activation. The plausibility of such a role for LTB4 is emphasized by our recent demonstration in macrophages that LTB4 elicits activation of p47phox via effects on the upstream kinase PKC- [31
].
In conclusion, our experiments show that n-6 PUFAs activate NADPH oxidase in glycogen-elicited PMNs, and this activation is partially dependent on 5-LO activity, LTB4 synthesis, and BLT1 signaling. Indeed, we demonstrated that LTB4 is required for two essential aspects of NADPH oxidase activation, namely, phosphorylation and translocation of p47phox, in PMNs stimulated by AA. This study presents a new model of NADPH oxidase activation by PUFAs, and the identification of a role for LTB4 suggests possible therapeutic interventions targeting this lipid mediator for inflammatory conditions in which ROI production is responsible for tissue injury.
Received October 14, 2004; revised May 26, 2005; accepted June 6, 2005.
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