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Am J Physiol Cell Physiol 294: C985-C993, 2008. First published February 20, 2008; doi:10.1152/ajpcell.00454.2007
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RECEPTORS AND SIGNAL TRANSDUCTION

Role of extracellular superoxide in neutrophil activation: interactions between xanthine oxidase and TLR4 induce proinflammatory cytokine production

Emmanuel Lorne,1,4 Jaroslaw W. Zmijewski,1,2 Xia Zhao,1 Gang Liu,1 Yuko Tsuruta,1 Young-Jun Park,1 Hervé Dupont,3 and Edward Abraham1

1Department of Medicine and 2Center for Free Radical Biology,University of Alabama at Birmingham, Birmingham, Alabama; 3Pole Anesthésie Réanimation du Centre Hospitalier Universitaire and 4Institut National de la Santé et de la Recherche Médicale, Amiens, France

Submitted 1 October 2007 ; accepted in final form 13 February 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reactive oxygen species (ROS) contribute to neutrophil activation and the development of acute inflammatory processes in which neutrophils play a central role. However, there is only limited information concerning the mechanisms through which extracellular ROS, and particularly cell membrane-impermeable species, such as superoxide, enhance the proinflammatory properties of neutrophils. To address this issue, neutrophils were exposed to superoxide generating combinations of xanthine oxidase and hypoxanthine or lumazine. Extracellular superoxide generation induced nuclear translocation of nuclear factor-{kappa}B (NF-{kappa}B) and increased neutrophil production of the NF-{kappa}B-dependent cytokines tumor necrosis factor-{alpha} (TNF-{alpha}) and macrophage inhibitory protein-2 (MIP-2). In contrast, there were no changes in TNF-{alpha} or MIP-2 expression when neutrophils lacking Toll-like receptor-4 (TLR4) were exposed to extracellular superoxide. Immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer (FRET) studies demonstrated association between TLR4 and xanthine oxidase. Exposure of neutrophils to heparin attenuated binding of xanthine oxidase to the cell surface as well as interactions with TLR4. Heparin also decreased xanthine oxidase-induced nuclear translocation of NF-{kappa}B as well as production of proinflammatory cytokines. These results demonstrate that extracellular superoxide has proinflammatory effects on neutrophils, predominantly acting through an TLR4-dependent mechanism that enhances nuclear translocation of NF-{kappa}B and increases expression of NF-{kappa}B-dependent cytokines.

reactive oxygen species; Toll-like receptor-4; nuclear factor-{kappa}b; signal transduction; heparin; glycosaminoglycans

UNDER PHYSIOLOGICAL CONDITIONS, reactive oxygen species (ROS) participate in intracellular signaling pathways, transcriptional regulation, and other cellular events involved in maintaining homeostasis (10). However, excessive generation of ROS or alterations in oxidant-antioxidant balance contribute to organ dysfunction in inflammatory conditions in which activated neutrophils play a central role, such as septic shock (7, 8), hemorrhage, or LPS-induced lung injury (21), intestinal ischemia-reperfusion (5, 6, 22), and chronic obstructive pulmonary disease (COPD) (3, 17, 19).

Alterations in the intracellular levels of specific ROS appear to have distinct effects on neutrophil function. For example, increased intracellular concentrations of superoxide result in activation of nuclear factor-{kappa}B (NF-{kappa}B) and enhanced proinflammatory cytokine production (23). In contrast, exposure of neutrophils to hydrogen peroxide, which can cross the cell membrane to increase intracellular hydrogen peroxide levels, results in inhibition of proteasomal activity and attenuation of I{kappa}B-{alpha} degradation upon Toll-like receptor-4 (TLR4) engagement, as well as decreased nuclear translocation of NF-{kappa}B- and LPS-induced proinflammatory cytokine production (44).

Unlike hydrogen peroxide, which is able to rapidly diffuse across cell membranes, superoxide, as a charged species, is unable to transit from extracellular to intracellular sites. In vivo studies have demonstrated that superoxide generation in the extracellular milieu is proinflammatory. For example, intestinal ischemia-reperfusion or large volume blood loss results in increased circulating concentrations of xanthine, hypoxanthine, xanthine oxidase, systemic production of superoxide, and the development of acute lung injury (4, 24, 29, 34). However, it has not been determined whether the proinflammatory effects associated with xanthine oxidase release are directly due to cellular interactions with superoxide, to increased production of hydrogen peroxide and other ROS that then affect cellular function, or to induction of secondary proinflammatory pathways initiated by cellular contact with xanthine oxidase.

The present studies were designed to examine the mechanisms by which extracellular superoxide results in neutrophil activation. We found that generation of superoxide through direct interaction of xanthine oxidase with TLR4 resulted in enhanced activation of NF-{kappa}B and production of NF-{kappa}B-dependent proinflammatory cytokines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents and antibodies. Thioglycolate, xanthine oxidase, hypoxanthine, lumazine, superoxide dismutase, catalase, and cytochrome c were purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640 was purchased from BioWhittaker (Walkersville, MD). FBS and penicillin-streptomycin were obtained from Gemini Bioproducts (Calabasas, CA). ELISA kits for TNF-{alpha} or MIP-2 were from R&D Systems (Minneapolis, MN). The antibodies to TLR4 (H-80), TLR5 (300 m), xanthine oxidase (T-17), and p65 (C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa 488-labeled anti-rabbit and Alexa 555-labeled anti-goat antibodies were purchased from Invitrogen (Eugene, OR). Custom antibodies for neutrophil isolation were purchased from StemCell Technologies (Vancouver, BC, Canada). Isoflurane was obtained from Abbott Laboratories (Chicago, IL).

Animals. Male C57/BL6, C57/10Scn (TLR4–/–), and C57/10J (TLR4+/+) mice, 10–12 wk of age, were purchased from Jackson Laboratory (Bar Harbor, ME) (28). The mice were kept on a 12-h light, 12-h dark cycle with free access to food and water. All experiments were conducted in accordance with institutional review board-approved protocols.

Neutrophil isolation and culture. Bone marrow neutrophils were isolated from the hips, femurs, and tibias of 8- to 12-wk-old mice and purified by negative selection using primary antibodies specific for the cell surface markers F4/80, CD4, CD45R, CD5, and TER119 StemCell Technologies as described previously (44). Neutrophil purity, as determined by Wright-Giemsa-stained cytospin preparations, was consistently >97%. Neutrophils were also isolated from the peritoneal cavity of mice 4 h after intraperitoneal injection with 2 ml of thioglycolate solution (3%). The peritoneal cell populations were consistently composed of >97% neutrophils. Neutrophils were cultured in RPMI 1640 medium containing FBS (0.5%) and treated as described in the figures. The cell viability was determined using a trypan blue staining and was consistently >95%.

Measurement of superoxide generated by hypoxanthine-xanthine oxidase system. The superoxide generated by hypoxanthine-xanthine oxidase system was measured in culture medium (RPMI-1640, 0.5% FBS) by using the superoxide-dependent reduction of cytochrome c assay (22). Briefly, the reaction was initiated in samples containing xanthine oxidase (1, 3, or 10 mU/ml), hypoxanthine (500 µM), and cytochrome c (10 µM), and the rate of cytochrome c reduction was recorded ({lambda} = 550 nm, {epsilon}M = 21 mM–1·cm–1) using a spectrophotometer (UV-2501PC Shimadzu; Shimadzu, Japan) for 10 min at 37°C. The concentration of superoxide was calculated by using a standard of reduced cytochrome c (Sigma), and superoxide production was corrected by subtraction of the superoxide dismutase (SOD, 256 mU/ml) insensitive rates. The rates of superoxide generated by 1, 3, or 10 mU/ml hypoxanthine-xanthine oxidase system were ~0.13, 0.51, or 0.92 nmol–1·min–1·ml–1, respectively.

Cytokine ELISA. TNF-{alpha} and macrophage inhibitory protein (MIP-2) were quantified using commercially available ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer's instructions and as described previously (38, 43, 44).

Nuclei isolation and Western blot analysis. Nuclear proteins were purified from 7 x 106 neutrophils as described previously (44). The protein concentration in the nuclear extracts was determined using Bradford reagent (Bio-Rad, Hercules, CA) with BSA as a standard. Samples were mixed with Laemmli sample buffer and boiled for 5 min. Equal amounts of proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Immobilon P, Millipore, Billerica, MA). The membranes were probed with specific antibodies to p65 (Santa Cruz, CA) followed by detection with horseradish peroxidase-conjugated goat anti-rabbit IgG. Bands were visualized by enhanced chemiluminescence (ECL plus, Amersham) and quantified by AlphaEaseFC Software (Alpha Innotech, San Leandro, CA). Each experiment was performed three or more times using cell populations obtained from separate groups of mice.

Electrophoretic mobility shift assay. Nuclear extracts were obtained from C57/10Scn (TLR4–/–) and C57/10J (TLR4+/+) bone marrow neutrophils after culture with or without xanthine oxidase. Electrophoretic mobility shift assay (EMSA) was performed as reported previously (35, 40, 42). In brief, the {kappa}B DNA sequence of the Ig gene was used. Synthetic double-stranded oligonucleotide probes for the {kappa}B site were filled in and were [32P]dATP end labeled (GE Healthcare) using Sequenase DNA polymerase: {kappa}B sequence, 5'-GCCATGGGGGGATCCCCGAAGTCC-3' (Geneka Biotechnology).

Immunoprecipitation assay (IP). TLR4+/+ and TLR4–/– bone marrow neutrophils (15 x 106) were incubated with xanthine oxidase (60 mU) for 15 min. TLR4 was then immunoprecipitated from the cell lysates, as described previously with minor modifications (26). In brief, neutrophils were lysed in immunoprecipitation (IP) buffer (20 mM Tris·HCl, pH 8.0, 137 mM NaCl, 2 mM EDTA, 5% glycerol, and 0.1% NP40) containing protease inhibitors (Roche, Mannheim, Germany). To remove insoluble particles, cell extracts were centrifuged (14,000 rpm) for 15 min at 4°C. TLR4 was immunoprecipitated by incubation of cell extracts (500 µg) with anti-TLR4 antibodies overnight at 4°C followed by incubation with recombinant protein A agarose (Invitrogen) for 2 h at 4°C. The immunoprecipitates were washed three times with IP buffer, boiled in x2 Laemmli buffer (40 µl), and then subjected to Western blot analysis.

Immunofluorescence microscopy and fluorescence resonance energy transfer analysis. Bone marrow neutrophils were cultured on coverslips in RPMI 1640 media supplemented with FBS (0.5%) and treated as indicated in the figure legends. Cells were then fixed with paraformaldehyde (4%) for 45 min. To detect cell membrane localization of xanthine oxidase, TLR4, or TLR5, samples were subsequently incubated with PBS containing 5% bovine serum albumin (BSA) for 30 min followed by incubation for 1 h at room temperature with antibodies specific for xanthine oxidase (T-17), TLR4 (H-80), or TLR5 (300 m). Coverslips were washed three times with PBS and then incubated with Alexa 488- and Alexa 555-labeled secondary antibodies for 1 h at room temperature. Antibodies were diluted 1:100 in PBS containing 5% BSA. The coverslips were mounted using a solution of PBS containing n-propyl gallate (0.2%) and glycerol (90%, vol/vol). Images were acquired by four bidirectional scans of the cells with the use of a Leica DMIRBE inverted epifluorescence/Nomarski microscope outfitted with Leica TCS NT laser confocal optics (absorption/emission filters: green 480/500 nm or red 561/570 nm). Images were merged and digital processing was performed using Adobe Photoshop (Adobe Systems, San Jose, CA) software.

Fluorescence resonance energy transfer (FRET) analysis (33) was used to measure proximity between TLR4 or TLR5 with xanthine oxidase bound to the cell surface. Samples for FRET analysis were prepared as for confocal microscopy, as described above. The baseline fluorescence (prebleach) of xanthine oxidase (Alexa 555) and TLR4 or TLR5 (Alexa 488) were measured using excitation/emission at 561/575-714 nm or 480/500-535 nm, respectively. Half of the cell was then exposed to high-energy excitation at 561 nm (Alexa 550, acceptor fluorochrome) to transfer energy to Alexa 488 (donor fluorochrome). After this bleaching process, emission intensity of the Alexa 555 and 488 fluorochromes was measured (postbleach). Bleaching of Alexa 555 was confirmed by being <30% of initial intensity. The increased fluorescence of Alexa 488 was determined by calculation of FRET efficiency using LEICA confocal software and formulas where the difference between values obtained from postbleach and prebleach were divided by the postbleach intensity. FRET efficiency from the nonexcited part of the cell was used to subtract the background. Data were averaged from at least 12 cells from three coverslips.

Statistical analysis. For each experiment, neutrophils were isolated and pooled from groups of mice (n = 3–4 mice in each group). One-way analysis of variance, the Tukey-Kramer Multiple Comparisons test (for multiple groups), or Student's t-test (for comparisons between two groups) were used. P < 0.05 was considered to be statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Xanthine oxidase-derived extracellular superoxide induces cytokine production by neutrophils. Exposure of bone marrow neutrophils to the superoxide generating combinations of xanthine oxidase and hypoxanthine or xanthine oxidase and lumazine (2,6-dihydroxypteridine) resulted in increased TNF-{alpha} and MIP-2 production in a dose-dependent manner (Fig. 1, A–D). Culture of peritoneal neutrophils with xanthine oxidase and hypoxanthine also resulted in increased generation of TNF-{alpha} and MIP-2 (Fig. 1, E and F). In these experiments, cellular viability was consistently >95% after 6 h of culture with all concentrations of xanthine oxidase (0, 3, and 10 mU/ml).


Figure 1
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  		Fig. 1. Incubation of neutrophils with xanthine oxidase (XO) induces cytokine production. Bone marrow (A–D) or peritoneal neutrophils (4 x 106/ml) (E and F) were cultured in RPMI 1640 media (0.5% FBS) with the indicated concentrations of XO and hypoxanthine (500 µM) (A, B, E, and F) or lumazine (500 µM) (C, D) for 5 h. Tumor necrosis factor-{alpha} (TNF-{alpha}) and macrophage inhibitory protein-2 (MIP-2) protein concentrations in the culture supernatants were measured using ELISA (n = 3, means ± SE, *P < 0.05 or **P < 0.01 compared with untreated). Two additional experiments demonstrated similar results.

 
Because NF-{kappa}B participates in the transcriptional regulation of TNF-{alpha} and MIP-2 (9, 11), we evaluated nuclear translocation of NF-{kappa}B in neutrophils cocultured with hypoxanthine and xanthine oxidase. As shown in Fig. 2, exposure of neutrophils to hypoxanthine-xanthine oxidase resulted in enhanced nuclear localization of the NF-{kappa}B p65 subunit in a dose-dependent manner (Fig. 2). The proinflammatory effects of the hypoxanthine-xanthine oxidase combination were directly due to the catalytic activity of xanthine oxidase since inclusion of allopurinol, a specific inhibitor of xanthine oxidase, into the neutrophil cultures completely inhibited generation of TNF-{alpha} and MIP-2 (Fig. 3, A and B).


Figure 2
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  		Fig. 2. Neutrophil exposure to XO induces nuclear translocation of nuclear factor-{kappa}B (NF-{kappa}B). Bone marrow neutrophils (4 x 106/ml) were cultured in 2 ml RPMI 1640 media (0.5% FBS) with hypoxanthine (500 µM) and XO (0, 1, 3, or 10 mU/ml) for 60 min. Nuclear extracts were then subjected to SDS-PAGE Western blot analysis with antibodies specific for p65. A representative Western blot shows hypoxanthine-XO-dependent p65 nuclear accumulation (inset), as well as densitometry determinations from 3 independent experiments (means ± SD, *P < 0.05 compared with control).

 

Figure 3
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  		Fig. 3. XO-derived superoxide, but not hydrogen peroxide, contributes to induction of TNF-{alpha} and MIP-2 production by neutrophils. A and B: XO (0 or 10 mU/ml) was preincubated with or without allopurinol (100 µM) for 4 h before addition to the bone marrow neutrophil cultures (4 x 106 cells/ml in RPMI 1640 media with 0.5% FBS) containing hypoxanthine (500 µM). Superoxide dismutase (SOD, 256 U/ml) (C and D) or catalase (CAT, 600 U/ml) (F and G) were added to the cultures (4 x 106 cells/ml in RPMI 1640 media, 0.5% FBS) 10 min before inclusion of XO (0 or 10 mU/ml). Cells were then incubated for 5 h. TNF-{alpha} and MIP-2 protein concentrations in culture supernatants were determined by ELISA (n = 3, means ± SE, *P < 0.05 or **P < 0.01 compared with XO-treated cells). E: SOD-dependent superoxide dismutation decreases in presence of neutrophils. The rate of XO (0.5 mU/ml) generated superoxide was determined in culture media supplemented with SOD (0 or 25 U/ml) in the presence or absence of neutrophils (2 x 105 cells/ml). Data are expressed as a percentage of SOD-dependent inhibition of the initial rate of superoxide production in presence or absence of neutrophils (n = 3, means ± SE, *P < 0.05 compared with XO without cells). Similar results were obtained from two additional experiments.

 
Catalytic oxidation of hypoxanthine or lumazine by xanthine oxidase produces superoxide and hydrogen peroxide (14). We therefore evaluated whether the proinflammatory effects of the hypoxanthine-xanthine oxidase and lumazine-xanthine oxidase combinations were due to superoxide or hydrogen peroxide. As shown in Fig. 3 (C–D and F–G), addition of superoxide dismutase, but not catalase, to the cultures decreased the effects of hypoxanthine-xanthine oxidase. Because inclusion of SOD was not able to completely reverse the proinflammatory effects of hypoxanthine-xanthine oxidase, we compared the ability of SOD to dismutate superoxide in the presence or absence of neutrophils. As shown in the Fig. 3E, SOD effectively dismutated hypoxanthine-xanthine oxidasegenerated superoxide (82%) in cultures that contained no neutrophils. However, there was significant decrease (nearly 35%) in SOD-dependent superoxide dismutation in the presence of cells, demonstrating that the ability of SOD to dismutate hypoxanthinexanthine oxidase-generated superoxide was less effective in the presence of neutrophils, presumably as a result of cell-bound xanthine oxidase being inaccessible to SOD. Taken together, these results indicate that generation of superoxide, rather than of hydrogen peroxide, is responsible for neutrophil activation after exposure to xanthine oxidase.

TLR4 participates in the activation of neutrophils by xanthine oxidase. Previous in vivo studies indicated that TLR4 was involved in hemorrhage-induced acute lung injury, a situation in which there are increased circulating concentrations of xanthine oxidase but no detectible LPS in plasma (4). Such findings suggested that interactions between TLR4 and reactive oxygen intermediates generated by xanthine oxidase might contribute to the production of proinflammatory cytokines in the lungs and the development of acute lung injury.

To examine the role of interactions between TLR4 and extracellular superoxide in cellular activation, neutrophils from TLR4–/– C57/10Scn (28) and control TLR4+/+ C57/10J mice were cultured with hypoxanthine-xanthine oxidase. As shown in Fig. 4, A–C, TNF-{alpha} and MIP-2 generation as well as NF-{kappa}B activation following neutrophil coculture with hypoxanthine and xanthine oxidase were significantly decreased in TLR4–/– compared with TLR4+/+ neutrophils.


Figure 4
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  		Fig. 4. Proinflammatory effects of XO are mediated by Toll-like receptor (TLR4). A and B: bone marrow neutrophils (4 x 106 cells/ml) isolated from TLR4–/– C57/10Scn or TLR4+/+ C57/10J mice were cultured in RPMI 1640 media (0.5% FBS) and hypoxanthine (500 µM) either with or without XO (10 mU/ml) for 5 h. TNF-{alpha} and MIP-2 protein concentrations in culture supernatants were measured by ELISA (n = 3, means ± SE, **P < 0.01 compared TLR4–/– to TLR4+/+). Two additional experiments demonstrated similar results. C: representative electrophoretic mobility shift assay shows that XO-induced NF-{kappa}B DNA binding activity was predominantly TLR4 dependent. Nuclear extracts were prepared from C57/10Scn (TLR4–/–) and C57/10J (TLR4+/+) bone marrow neutrophils (4 x 106/ml cells) cultured in 2 ml of RPMI 1640 media (0.5% FBS) and treated with XO (0 or 10 mU/ml) for 90 min. Two additional experiments provided similar results. D: interaction of TLR4 and XO in bone marrow neutrophils. Western blot analysis shows purified bovine XO (30 or 0 ng), XO coimmunoprecipitated using anti-TLR4-specific antibodies (IP), and XO levels in cell extracts prepared from TLR4+/+ and TLR4–/– neutrophils, respectively.

 
Because neutrophil activation by xanthine oxidase was TLR4 dependent, we examined if xanthine oxidase could directly associate with TLR4. Using coimmunoprecipitation, we found interaction between TLR4 and xanthine oxidase, in TLR4+/+ neutrophils (Fig. 4D). Confocal microscopy and FRET analysis were used to confirm membrane-based interaction between xanthine oxidase and TLR4 on neutrophils. Initial studies tested the specificity of the TLR4 antibody on neutrophils from TLR4–/– (C57BL/10Scn) and control TLR4+/+ (C57BL/10J) mice. Whereas no fluorescence was observed with C57BL/10Scn neutrophils, positive membrane staining for TLR4 was found with C57/BL6 neutrophils (Fig. 5A).


Figure 5
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  		Fig. 5. XO and TLR4 colocalize on the neutrophil surface. TLR4+/+ or TLR4–/– bone marrow neutrophils (4 x 106 cells/ml) cultured in RPMI 1640 media (0.5% FBS) were treated with XO (0 or 3 mU/ml) for 60 min and then stained with TLR4 or TLR5 and XO-specific antibodies followed by image acquisition using confocal microscopy. A–C: representative images showing TLR4 or TLR5 (green), XO (red), and merge indicates colocalization (yellow) of TLR4 and XO but not of TLR5 and XO. The areas of interest (insets in B and C, white border) are magnified and shown on the right side of each image. C: fluorescence resonance energy transfer (FRET) acceptor bleaching. Representative images show donor prebleach (TLR4, green), acceptor prebleach (XO, red), acceptor bleaching (XO, red), and the FRET signal. Regions of interest (inset border) indicate the area exposed to high-energy excitation. D: mean FRET efficiency in neutrophils treated as described above (n = 12, means ± SE, *P < 0.05 compared with treated cells).

 
Confocal imaging of TLR4+/+ neutrophils revealed a punctative, colocalized pattern of TLR4 and xanthine oxidase on the cell surface membrane (Fig. 5B). Although xanthine oxidase was found to be associated with the cell surface of TLR4–/– neutrophils, the pattern of xanthine oxidase staining was diffuse compared with the focal localization present on TLR4+/+ neutrophils (Fig. 5, A and B). In contrast to colocalization of TLR4 and xanthine oxidase, there was no association between TLR5 and xanthine oxidase (Fig. 5C). Figure 5, D–E, shows FRET analysis that confirms interaction between TLR4 and xanthine oxidase but no evidence of similar colocalization of TLR5 and xanthine oxidase.

Effects of heparin on membrane-bound xanthine oxidase and xanthine oxidase-mediated cytokine production by neutrophils. Xanthine oxidase is known to bind to glycosaminoglycans in the extracellular matrix (2, 30). Heparin has been shown to compete with xanthine oxidase for binding to glycosaminoglycans (2, 30, 31). Under in vivo conditions, intravascular injection of heparin releases xanthine oxidase from endothelial cells and leads to an increase in circulating concentrations of xanthine oxidase (2).

To test whether heparin potentially releases membrane-bound xanthine oxidase from neutrophils, cells were incubated with xanthine oxidase in the presence or absence of heparin, and the activity of xanthine oxidase in supernatants was determined using the cytochrome c reduction assay. In the absence of cells, addition of heparin to culture media had no effect on the rate of superoxide production by the combination of xanthine oxidase and hypoxanthine (Fig. 6A). In contrast, the rate of superoxide production was increased in the supernatants collected from neutrophils coincubated with heparin (Fig. 6B). Moreover, addition of heparin to neutrophils cocultured with hypoxanthine-xanthine oxidase resulted in decreased nuclear translocation of NF-{kappa}B (Fig. 6, E and F) as well as diminished production of TNF-{alpha} and MIP-2 compared with that induced by hypoxanthine-xanthine oxidase alone (Fig. 6, C and D).


Figure 6
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  		Fig. 6. Effects of heparin on membrane-associated XO and XO-induced cytokine production by neutrophils. A: superoxide generation by hypoxanthine (500 µM) and XO (10 mU/ml) in cell culture media alone was measured in the presence or absence of heparin (5 U/ml). B: bone marrow neutrophils (4 x 106 cells/ml) cultured in RPMI 1640 media (0.5% FBS) with XO (10 mU/ml) in the presence or absence of heparin for 60 min at 37°C. Rates of superoxide generation in the supernatants were then determined using the cytochrome c reduction assay (n = 3, means ± SD, *P < 0.05 compared with cells treated with XO only). C and D: heparin inhibits XO-mediated cytokine production by neutrophils. Bone marrow neutrophils (4 x 106 cells/ml) cultured in RPMI 1640 media (0.5% FBS) with hypoxanthine (500 µM) in the presence or absence of heparin (1 U/ml) and XO (0 or 3 mU/ml) for 5 h. TNF-{alpha} and MIP-2 protein concentrations in culture supernatants were determined using ELISA (n = 3, means ± SD, *P < 0.05 compared with cells treated with XO only). E and F: heparin inhibits XO-induced p65 nuclear accumulation. Bone marrow neutrophils (4 x 106/ml) cultured in 2 ml in RPMI 1640 media (0.5% FBS) with heparin (0 or 1 U/ml), hypoxanthine (500 µM), and XO (0 or 3 mU/ml) for 60 min. A representative Western blot is shown as well as mean values from 3 independent experiments (means ± SD, **P < 0.01).

 
The results of the above experiments suggested that heparin might affect the association of xanthine oxidase with the neutrophil surface. Confocal microscopy and FRET analysis confirmed this hypothesis by demonstrating that heparin exposure decreased the fluorescence of xanthine oxidase on the cell surface membrane (Fig. 7A) and also decreased FRET efficiency between TLR4 and xanthine oxidase (Fig. 7B).


Figure 7
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  		Fig. 7. Heparin inhibits association of XO and TLR4 on the neutrophil cell surface. A: representative images show cell membrane localization of TLR4 and XO. Bone marrow neutrophils preincubated with heparin (0 or 1 U/ml) for 30 min were treated with XO (3 mU/ml) for 1 h. Cells were then stained with TLR4 and XO-specific antibodies followed by image acquisition using confocal microscopy. The areas of interest (insets, white border) are magnified and shown on the right side of each image. XO, red; TLR4, green; and merged images are shown. B: mean fluorescence resonance energy transfer (FRET) efficiency in neutrophils treated as described above (n = 12, means ± SE, *P < 0.05 compared with treated cells).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present studies, extracellular superoxide potently induced nuclear translocation of NF-{kappa}B as well as the production of proinflammatory cytokines in neutrophils. The mechanism for the proinflammatory effects of superoxide appeared to be through association of xanthine oxidase with TLR4. In particular, confocal microscopy and FRET studies demonstrated direct interaction between TLR4 and xanthine oxidase on the cell surface, and neutrophil activation by exposure to the superoxide generating combination of hypoxanthine-xanthine oxidase was decreased to near baseline levels in TLR4-deficient neutrophils.

In previous experiments, we found that incubation of neutrophils with paraquat, an intracellular generator of superoxide, increased NF-{kappa}B activation and proinflammatory cytokine secretion (23). Paraquat also enhanced TLR4-mediated nuclear translocation of NF-{kappa}B and expression of NF-{kappa}B-dependent cytokines, such as TNF-{alpha} and MIP-2, consistent with a proinflammatory effect for intracellularly generated superoxide in neutrophils (23). However, the effects of extracellular superoxide on neutrophil activation have not been previously reported. In vivo studies indicated that increased extracellular superoxide production accompanies hemorrhage-induced acute lung injury, a condition in which activated neutrophils play a central role and circulating concentrations of xanthine oxidase, but not LPS, are increased (4, 32).

In vitro studies, from our laboratory and others, have shown that cellular exposure to the superoxide-generating combination of xanthine-xanthine oxidase could induce NF-{kappa}B activation in splenocytes, NF-{kappa}B-dependent cytokine generation in smooth muscle cells and monocytes, as well as enhanced NF-{kappa}B/DNA binding activity in podocytes (12, 18, 20, 25, 35). Because superoxide is unable to diffuse across membranes, we did not expect that extracellular superoxide could directly affect intracellular signaling pathways. The present experiments confirmed this hypothesis by demonstrating that the mechanism through which extracellular superoxide induced neutrophil activation was by association with cell surface TLR4 and activation of TLR4-linked pathways that lead to activation of NF-{kappa}B.

The association of xanthine oxidase with TLR4 on the neutrophil membrane appears to occur through binding to glycosaminoglycans, as shown by reduction of FRET efficiency between xanthine oxidase and TLR4 when neutrophils were preincubated with heparin. Such interactions between xanthine oxidase and glycosaminoglycans on the cell surface also provide an explanation for the inability of allopurinol, when added to the cultures, to suppress completely hypoxanthine-xanthine oxidase-induced neutrophil activation. In particular, binding of xanthine oxidase to glycosaminoglycans has been shown to limit inhibition of xanthine oxidase activity by oxipurinol (16). In addition, there is diminished ability of allopurinol to block the catalytic, superoxide-producing activity of endothelial cell bound xanthine oxidase (15). In the present experiments, incubating xanthine oxidase with allopurinol for 4 h before addition to cell cultures completely prevented its ability to stimulate neutrophil activation. In contrast, addition of allopurinol with or immediately after inclusion of xanthine oxidase in the cell cultures was unable to affect xanthine oxidase-induced increases of NF-{kappa}B activation and cytokine production (data not shown). These observations, which are consistent with membrane-associated xanthine oxidase being relatively inaccessible to allopurinol, could provide at least a partial explanation for lack of efficacy of allopurinol in decreasing organ dysfunction after severe accidental trauma, a situation associated with increased circulating, and presumably cell-bound concentrations of xanthine oxidase (13, 39).

In these studies, we used CuZn-superoxide dismutase and catalase to determine the specific roles of superoxide and hydrogen peroxide in xanthine oxidase-induced neutrophil activation. Other laboratories have shown that the binding of xanthine oxidase to endothelial cells decreases the inhibitory efficiency of CuZn-SOD so that it is only able to diminish xanthine oxidase-induced superoxide generation by about 30% (30). Such findings suggested that glycosaminoglycan-bound xanthine oxidase partitions into a SOD-resistant compartment (15, 30). Consistent with such previous findings, we found that CuZn-SOD was less efficient in decreasing xanthine oxidase-induced superoxide generation in the presence of neutrophils compared with conditions where neutrophils were absent. Similarly, CuZn-SOD significantly decreased, but did not eliminate, the effects of xanthine oxidase on cytokine generation by neutrophils. Of note, no additional inhibitory effects of CuZn-SOD on cytokine production by hypoxanthine-xanthine oxidase-stimulated neutrophils were found even when the concentration of Cu-Zn SOD in the culture media was increased to 500 U/ml (data not shown). Catalase did not affect cytokine production by neutrophils coincubated with hypoxanthine-xanthine oxidase, indicating that hydrogen peroxide does not contribute to the potentiating effects of xanthine oxidase. These results are consistent with our previous studies that found no effect of catalase on xanthine-xanthine oxidase-induced NF-{kappa}B activation in splenocytes (35). Of note, incubation of neutrophils with catalase produced enhanced production of TNF-{alpha} and MIP-2. These findings are similar to those reported in our previous study that found a two- to threefold increase in TNF-{alpha} and MIP-2 by catalase-treated neutrophils (44). These effects of catalase on the production of NF-{kappa}B-dependent cytokines are likely to result from enhanced proteasomal-associated degradation of I{kappa}B-{alpha} as a result of diminished intracellular levels of hydrogen peroxide. In particular, our previous studies (44) showed that hydrogen peroxide had inhibitory effects on proteasomal function and degradation of I{kappa}B-{alpha}, both in LPS-treated and resting neutrophils; treatment of cells with catalase, which results in decreased intracellular concentrations of hydrogen peroxide, therefore would be expected to be associated with enhanced proteasomal degradation of I{kappa}B-{alpha} and increased activation of NF-{kappa}B.

The present experiments demonstrate that TLR4 is centrally involved in mediating the proinflammatory effects of extracellular superoxide. Such results are consistent with previous studies indicating a role for TLR4 in oxidant-induced organ dysfunction. For example, TLR4 was shown to participate in hemorrhage-induced neutrophil activation and increases in pulmonary concentrations of TNF-{alpha} and the development of acute lung injury, a situation in which circulating concentrations of xanthine oxidase and production of ROS are increased, even though there is no detectible LPS in plasma (4). In vivo studies demonstrated that therapy with eritoran, a specific TLR4-MD2 inhibitor, attenuated myocardial ischemia-reperfusion injury (36). Similarly, infarction size after coronary ligation was decreased in C3H/HeJ mice that express a nonfunctional TLR4 when compared with that of control C3H/HeN mice (37).

In the present experiments, we found that xanthine oxidase colocalizes and interacts with TLR4 on the cell surface of neutrophils. The association of xanthine oxidase and TLR4 provides a direct mechanism whereby xanthine oxidase-mediated superoxide production can affect TLR4-related signaling events, especially given the extremely short half-time of superoxide, before dismutation to hydrogen peroxide occurs. However, despite the evidence provided in this study that xanthine oxidase-derived superoxide activates neutrophils, the actual mechanism through which superoxide affects TLR4 signaling, including the extracellular domain of TLR4 involved, remains an important question for future investigation.

This study is the first to show direct binding of xanthine oxidase to neutrophils via a glycosaminoglycan-mediated mechanism. Binding of xanthine oxidase to the surface of endothelial cells is also mediated by glycosaminoglycans and is reversible by cell exposure to heparin (2, 30, 31). Addition of heparin to endothelial cells in vitro releases free xanthine oxidase into the culture media (2, 30). Similarly, injection of human subjects with heparin results in increased plasma concentrations of xanthine oxidase (2). In the present experiments, we found that heparin decreases the binding of xanthine oxidase to neutrophils and also diminishes xanthine oxidase-mediated cytokine production. Confocal microscopy and FRET analysis demonstrated that addition of heparin to xanthine oxidase-exposed neutrophils decreased the interaction between TLR4 and xanthine oxidase, thus providing a mechanism for the inhibitory effects of heparin on xanthine oxidase-induced cellular activation. Such findings suggest that therapy with heparin may be beneficial in diminishing the severity of acute inflammatory responses in pathophysiological conditions, such as sepsis and acute lung injury, in which xanthine oxidase and neutrophils play a contributory role. Of note, several analyses of large cohorts of septic patients have indicated that heparin administration is associated with reduced mortality (1, 27, 41). The present studies suggest that a potential mechanism for this beneficial effect on patient outcome is through reducing xanthine oxidase-induced TLR4-dependent neutrophil activation.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health Grants HL-62221, HL-76206, and HL-068743 (to E. Abraham) and by the Société Française d'Anesthésie et de Réanimation and the University Hospital of Amiens (France) (to E. Lorne).


   ACKNOWLEDGMENTS
 
We acknowledge Albert Tousson (High Resolution Imaging Facility at UAB) for his help during FRET analysis and Youhong Zhang for his technichal support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Abraham, Dept. of Medicine, Univ. of Alabama at Birmingham, BDB 420, 1530 3rd Ave., S, Birmingham, AL 35294-0012 (e-mail: eabraham{at}uab.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 GRANTS
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