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Originally published online as doi:10.1189/jlb.0705411 on April 19, 2006

Published online before print April 19, 2006
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(Journal of Leukocyte Biology. 2006;80:152-163.)
© 2006 by Society for Leukocyte Biology

Modulation of IgE-dependent COX-2 gene expression by reactive oxygen species in human neutrophils

Antonio Vega*,{dagger}, Pedro Chacón*,{dagger}, Gonzalo Alba*, Rajaa El Bekay*,{ddagger}, Javier Monteseirín*,{ddagger}, José Martín-Nieto§ and Francisco Sobrino*,1

* Departamento de Bioquímica Médica y Biología Molecular, Universidad de Sevilla, Sevilla, Spain;
{dagger} Servicio de Inmunología y Alergia, Hospital Universitario Virgen Macarena, Sevilla, Spain;
{ddagger} Clínica Sagrado Corazón, Sevilla, Spain; and
§ Departamento de Fisiología, Genética y Microbiología, Universidad de Alicante, Alicante, Spain

1 Correspondence: Dpto. Bioquímica Médica y Biología Molecular, Facultad de Medicina, Universidad de Sevilla, E-41009 Sevilla, Spain. E-mail: fsobrino{at}us.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cyclooxygenase (COX) is a key enzyme in prostaglandin (PG) synthesis. Up-regulation of its COX-2 isoform is responsible for the increased PG release, taking place under inflammatory conditions, and also, is thought to be involved in allergic and inflammatory diseases. In the present work, we demonstrate that COX-2 expression becomes highly induced by anti-immunoglobulin E (IgE) antibodies and by antigens in human neutrophils from allergic patients. This induction was detected at mRNA and protein levels and was accompanied by a concomitant PGE2 and thromboxane A2 release. We also show evidence that inhibitors of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, such as 4-(2-aminoethyl)benzenesulphonyl fluoride and 4-hydroxy-3-methoxyaceto-phenone, completely cancelled anti-IgE-induced COX-2 protein up-regulation, suggesting that this process is mediated by reactive oxygen species (ROS) derived from NADPH oxidase activity. Moreover, the mitogen-activated protein kinases (MAPKs), p38 and extracellular signal-regulated kinase, and also, the transcription factor, nuclear factor (NF)-{kappa}B, are involved in the up-regulation of COX-2 expression, as specific chemical inhibitors of these two kinases, such as SB203580 and PD098059, and of the NF-{kappa}B pathway, such as N({alpha})-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal, abolished IgE-dependent COX-2 induction. Evidence is also presented, using Fe2+/Cu2+ ions, that hydroxyl radicals generated from hydrogen peroxide through Fenton reactions could constitute candidate modulators able to directly trigger anti-IgE-elicited COX-2 expression through MAPK and NF-{kappa}B pathways. Present results underscore a new role for ROS as second messengers in the modulation of COX-2 expression by human neutrophils in allergic conditions.

Key Words: prostaglandins • allergy • signal transduction • ROS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostaglandins (PGs) play important roles in many biological processes, including homeostatic and immune responses [1 ], and an overproduction of arachidonic acid (AA) metabolites is associated with chronic inflammation and allergic diseases [2 , 3 ]. PGH2 synthase [cyclooxygenase (COX)] is the rate-limiting enzyme in the conversion of AA into eicosanoids, i.e., PGs and thromboxanes (TXs) [1 ]. COX exists in two isoforms, COX-1 and COX-2, of which COX-1 is constitutively expressed in most tissues and appears responsible for the production of PGs under normal, physiological conditions [2 , 3 ], and COX-2 expression is associated with inflammation [2 , 3 ] and becomes induced by various proinflammatory stimuli, including mitogens, bacterial lipopolysaccharide (LPS), and cytokines [2 3 4 ].

Reactive oxygen species (ROS) are highly diffusible, ubiquitous ions and radicals generated from the reduction of molecular oxygen. They include species such as superoxide anions (O2·–), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH). ROS are produced during the respiratory burst of neutrophils as a normal defense mechanism against pathogens. They modulate, however, multiple cellular functions such as cell growth and differentiation, proliferation, apoptosis, and gene expression, acting on transductional and transcriptional regulatory pathways [5 ]. The most important source contributing to ROS generation in neutrophils is the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system [6 ], which is composed of various subunits distributed between the plasma membrane (gp91phox and p22phox) and the cytosol (p47phox and p67phox) in resting phagocytic cells. When these become activated, the NADPH oxidase cytosolic subunits associate with membrane-bound, inactive components and assemble into a catalytically active enzyme [6 ].

The molecular mechanisms whereby COX-2 expression becomes induced remain unclear. Recent studies have highlighted a connection between up-regulation of COX-2 expression and the generation of ROS during monocyte-to-macrophage differentiation and the activation of mitogen-activated protein kinase (MAPK)-dependent pathways [7 ]. The interplay between MAPK-dependent pathways, increase of ROS levels, and activation of the transcription factor nuclear factor (NF)-{kappa}B is largely known in neutrophils [8 , 9 ]. In this context, COX-2 expression at the mRNA level is regulated positively through the binding of NF-{kappa}B and other transcription factors, such as the nuclear factor of activated T cells, the CCAAT enhancer-binding protein, or the cyclic adenosine monophosphate-responsive element-binding protein, to cis-acting, regulatory elements present in the COX-2 promoter [10 11 12 ].

The capacity of human neutrophils to induce COX-2 expression under inflammatory conditions has been shown previously [4 ], and there is ample evidence of the participation of this cell type in allergic processes [13 14 15 ]. In this regard, we have shown previously that a number of antigens are able to specifically activate the respiratory burst in neutrophils isolated from allergic patients sensitized to the same such antigens [16 ]. With this background, the present work was undertaken to assess whether in human neutrophils from allergic patients, COX-2 expression becomes induced upon their challenge with specific antigens or with anti-immunoglobulin E (IgE) antibodies and also to analyze the potential involvement of ROS and NADPH oxidase in this process. We here provide evidence that ROS-dependent signaling through the MAPK and NF-{kappa}B pathways, driven by NADPH oxidase, represents a mechanism previously unrecognized for the regulation of COX-2 expression upon neutrophil challenge with antigens or anti-IgE molecules. This finding highlights the possibility that unquenched ROS could actively contribute to the development of allergic inflammation by acting as second messengers in the modulation of PGE2 and TXA2 synthesis through COX-2 up-regulation by human neutrophils.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and antibodies
Antigens were commercially available extracts, including D1 (from Dermatophagoides pteronyssinus), G3 (from Dactylis glomerata), T9 (from Olea europaea), E2 (from dog epithelium), and M6 (from Alternaria alternata), purchased from Bial-Arístegui (Bilbao, Spain). Goat anti-human IgE antibody, goat anti-human IgG antibody, and the Perm-cell kit were from Caltag Laboratories (Burlingame, CA). H2O2, Escherichia coli LPS, FeSO4, CuSO4, phorbol 12-myristate-13-acetate (PMA), goat IgG, N({alpha})-benzyloxycarbonyl-l-leucyl-l-leucyl-l-leucinal (MG-132), 4-hydroxy-3-methoxyaceto-phenone (HMAP), 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), 2-amino-1,2,4-triazole, and 1,10-phenanthroline (PHE) were from Sigma-Aldrich (Madrid, Spain). PD098059 and SB203580 were products of Calbiochem (Madrid, Spain). [{gamma}-32P]Adenosine 5'-triphosphate (ATP; specific activity, 3000 Ci/mmol) and polymerase chain reaction (PCR) primers were from Amersham-Pharmacia-Biotech (Barcelona, Spain). Random primers were obtained from Roche (Madrid, Spain). The double-stranded oligonucleotide 5'-AGTGAGGGGACTTTCCCAGGC-3', containing a consensus NF-{kappa}B site (underlined), and Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) were obtained from Promega (Madison, WI). Unconjugated mouse monoclonal antibodies (mAb) to human COX-2 were obtained from Cayman Chemical (Ann Arbor, MI). Mouse mAb against human ß-actin (sc-8432), rabbit polyclonal antibody against human inhibitor of {kappa}B (I-{kappa}B; {alpha} isoform; sc-371), mouse antiphosphospecific Jun N-terminal kinase (JNK)1/2 (Thr183/Tyr185) antibody (sc-6254), and antibodies recognizing total (unphosphorylated plus phosphorylated) JNK1/2 (sc-7345) were purchased from Santa Cruz Biotechnology (CA). The rabbit polyclonal antiphosphospecific p38MAPK (Thr180/Tyr182), mouse monoclonal antiphosphospecific extracellular signal-regulated kinase (ERK)1/2 (Thr202/Tyr204), and antibodies recognizing the total forms of these two MAPKs were from New England Biolabs (Beverly, MA). Mouse mAb against CD9 and CD203 were from Immunotech-IZASA (Barcelona, Spain). Goat anti-mouse IgG-coated micromagnetic beads were from Miltenyi Biotec (Bergisch-Gladbach, Germany). Antisera against p47phox and p67phox were kindly donated by Thomas L. Leto (National Institutes of Health, Bethesda, MD). All culture reagents had endotoxin levels of ≤0.01 ng/ml, as tested by the Coatest Limulus lysate assay (Chromogenix, Mölndal, Sweden).

Patients and controls
The groups studied included adult, atopic patients with bronchial asthma and healthy, nonatopic volunteer controls. The asthmatic patients had given positive results on skin-prick (Bial-Arístegui) and specific IgE tests (HYTEC 288, Hycor Biomedical-IZASA, Barcelona, Spain) to at least one common allergenic antigen. These subjects had not received any treatment or specific hyposensitization and had not experienced episodes of asthma for at least 3 months or respiratory-tract infections in the 4 weeks before blood sampling. The healthy group had no history of allergy or bronchial symptoms and was negative for the skin-prick and specific IgE tests toward a battery of inhalant allergenic antigens (house dust mites, pollens, molds, and animal danders). The Universidad de Sevilla Ethics Committee (Spain) approved this study previously, and each subject had given prior informed consent.

Neutrophil isolation and cell culture conditions
Human peripheral neutrophils were isolated as described previously [17 ]. For further purification, neutrophil preparations were incubated with mouse anti-human CD9 and CD203 antibodies and goat anti-mouse IgG-coated micromagnetic beads. The purity of neutrophils was on average >99% (<0.1% eosinophil and close to 0% basophil contamination) and was used immediately after isolation. Neutrophils were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and maintained at 37°C in an atmosphere of 5% CO2 and 95% O2. None of the reagents used in this work significantly affected the viability of the cells at the concentrations used, as confirmed by the trypan blue dye exclusion test. Furthermore, after incubation with anti-IgE antibodies for 24 h, neutrophil viability was found to be >98% by measuring lactate dehydrogenase activity [18 ].

RT-PCR analysis of COX-2 mRNA levels
Total RNA from cultured neutrophils (107 cells) was isolated using the guanidine-phenol method [19 ], and 1–2 µg RNA was reverse-transcribed into cDNA using M-MLV RT and random primers. cDNA was amplified by PCR using the following specific primers for COX-2 (GenBank Accession Number M90100) or glyceraldehyde 3-phosphaste dehydrogenase (GAPDH; GenBank Accession Number J04038) as a house-keeping gene control: 5'-TTCAAATGAGATTGTGGGAAAATTGCT-3' (COX-2 sense), 5'-AGATCATCTCTGCCTGAGTATCTT-3' (COX-2 antisense), 5'-CCACCCATGGCAAATTCCATGGCA-3' (GAPDH sense), 5'-TCTAGACGGCAGGTCAGGTCCACC-3' (GAPDH antisense). The reaction was performed by 35 cycles each of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. Amplified PCR products were of 300 pb for COX-2 gene and of 605 pb for GAPDH gene.

Western blot analysis of COX-2 protein expression
Neutrophils (107 cells) were washed with phosphate-buffered saline (PBS) and lysed by incubation for 30 min in a buffer composed of 20 mM Hepes, pH 7.9, 5 mM KCl, 0.1% Nonidet P-40 (NP-40), 1 mM EDTA, 1 mM dithiothreitol (DTT), and 10 mM NaF, supplemented with the following protease-inhibitor mixture: 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml soybean trypsin inhibitor, 10 µg/ml N-tosyl-L-phenylalanyl-chloromethyl ketone, 10 µg/ml captopril, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM benzamidine, and 10 mM iodoacetamide. After centrifugation at 14,000 g, proteins (80 µg) were separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes as described [20 ]. These were probed for 1 h without need of prior blocking with specific antibodies against human COX-2 or ß-actin (1:2000 dilution) as a control of even protein loading. Primary antibody binding was detected by using horseradish peroxidase (HRP)-conjugated anti-rabbit or anti-mouse IgG antibodies (1:5000 dilution) followed by enhanced chemiluminescence (ECL) [21 ].

p47phox and p67phox membrane translocation
Membrane and cytosolic fractions were extracted from neutrophils as described previously [22 ]. Proteins were subjected to SDS-PAGE, electroblotted onto PVDF membranes as previouly described [20 ], and incubated for 1 h with goat anti-p47phox or anti-p67phox IgG antibodies as above. Antibody binding was detected by incubation with HRP-conjugated anti-goat IgG antibodies (1:5000 dilution) followed by ECL. Protein levels (membrane translocation and cytosol disappearance) were determined by scanning densitometry analysis using the Scion Image software (Scion Corp., Frederick, MD) and are presented in arbitrary units.

p38MAPK, ERK1/2, and JNK1/2 phosphorylation
Neutrophils (107 cells) were preincubated for 18 h in RPMI medium at 37°C to reduce the p38MAPK, JNK1/2, and ERK1/2 basal phosphorylation levels usually found in our preparations of human neutrophils. These were then subjected to the treatments indicated in each case, washed with PBS, and immediately lysed by incubation for 30 min in a buffer composed of 20 mM Tris-HCl, pH 7.4, 50 mM ß-mercaptoethanol, 2 mM EDTA, 25 mM NaF, 1% NP-40, 1% Triton X-100, 2 mM diisopropylfluorophosphate (DFP), and the protease-inhibitor mixture above. Protein extracts (80 µg) were resolved on 10% SDS-PAGE gels and electrotransferred to PVDF membranes, which were probed for 1 h with antiphospho-p38MAPK, antiphospho-ERK1/2, or antiphospho-JNK1/2 antibodies (1:2000 dilution). Antibody binding was detected by using HRP-conjugated anti-rabbit or anti-mouse IgG antibodies (1:5000 dilution) followed by ECL.

PGE2 release
Neutrophils (107 cells) were incubated in 1.5 ml RPMI medium on 24-well plates in the presence of antigen or anti-IgE antibody at the doses indicated in each case. The concentrations of PGE2 released were measured spectrophotometrically in the culture supernatants after neutrophil removal by centrifugation at 14,000 g for 5 min by using the PGE2 enzyme immunoassay kit (Cayman Chemical) according to the manufacturer’s protocol.

Measurement of TXA2 production
Neutrophils (107 cells) were incubated in 1.5 ml RPMI medium on 24-well plates as described above for PGE2 release, and TXA2 was assayed after neutrophil removal by centrifugation. As TXA2 has a short half-life (37 s) and is rapidly hydrolyzed nonenzymatically to its stable derivative TXB2, the Thromboxane B2 enzyme immunoassay kit (Cayman Chemical) was used to measure free TXA2 indirectly.

ROS levels
The levels of ROS were analyzed by luminol-amplified chemiluminescence, measured in a BioOrbit 1250 luminometer (Turku, Finland) at 37°C, basically as described [16 ]. Briefly, neutrophils (106 cells) were incubated for 5 min in 1 ml PBS supplemented with 10 mM glucose, 500 µM CaCl2, 5 µM luminol, and 100 µM sodium azide for 5 min. Then, allergens or anti-IgE antibodies as stimulants were added at the doses indicated in each case. The chemiluminescent response was measured every 1 min in each reaction and was expressed as the peak of luminescence recorded at 10 min.

I-{kappa}B cytosolic levels
For immunoblot analysis of I-{kappa}B-{alpha} levels, the cytoplasmic fraction was obtained from neutrophils as described previously [21 ] and subjected to SDS-PAGE, electroblotting onto PVDF membranes. These were probed with rabbit anti-human I-{kappa}B-{alpha} (1:2000 dilution) and thereafter, with HRP-conjugated anti-rabbit IgG (1:5000 dilution).

Electrophoretic mobility shift assay (EMSA) of NF-{kappa}B DNA-binding activity
Nuclear extracts were obtained as described previously [21 ] with minor modifications. The nuclear pellet was resuspended in 25 µl ice-cold relaxation buffer, additionally containing 10% (v/v) glycerol and 380 mM NaCl, supplemented with an antiprotease mixture composed of 2 mM DFP, 1 mM PMSF, 10 mM iodoacetamide, 1 mM benzamidine, and 10 µg/ml each aprotinin, leupeptin, and captopril. The double-stranded NF-{kappa}B oligonucleotide was end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase. Nuclear proteins (10 µg) were incubated with radioactive probes for 30 min on ice in binding buffer (20 mM Hepes, pH 7.8, 6% glycerol, 1 mM EDTA, 50 mM KCl, 1 mM DTT, and 0.1% NP-40), supplemented with 1 µg poly-(dI-dC) and 0.1 µg poly-L-lysine. Protein-DNA complexes were resolved in native 6% polyacrylamide gels and visualized by autoradiography. For DNA-competition assays, a 100-fold molar excess of unlabeled oligonucleotide was included in the reactions prior to addition of the radiolabeled probe.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of COX-2 expression by specific antigens and anti-IgE antbody in human neutrophils
To evaluate the potential role of antigens as inducers of COX-2 expression, neutrophils, isolated from a patient specifically sensitized to the G3 antigen, were cultured in the presence of this antigen, and the expression of the COX-2 enzyme was analyzed by immunoblotting. Figure 1 shows that whereas COX-2 was essentially undetectable in untreated neutrophils, after cell stimulation with the G3 antigen, expression of this protein was induced in a dose- and a time-dependent manner. Maximum COX-2 levels were attained at a 10-µg/ml concentration of the G3 antigen (Fig. 1A) , a dose at which the COX-2 protein was detectable after 6 h of treatment, and the peak of expression was reached at 12–24 h (Fig. 1C) . Neutrophil samples from patients allergic to other antigens gave a similar response toward their specific sensitizing antigens (data not shown). Further studies were addressed to analyze whether anti-human IgE antibodies could also induce COX-2 expression in allergic patients. Results similar to those obtained for the antigens above were obtained with anti-IgE antibodies used as the stimulus in neutrophils from a G3-allergic patient (Fig. 1B and 1D) . Figure 1E illustrates that in neutrophils from a patient exhibiting high serum levels of IgE specific for M6, T9, and G3, COX-2 synthesis became induced when they were cultured in the presence of either of these three antigens. Conversely, when neutrophils were cultured with antigens to which the patient was not sensitized, such as E1, D1, or D2, the COX-2 protein was not detected (Fig. 1E) . The specificity of this response was assessed further in neutrophils from healthy subjects, in which COX-2 expression was not elicited by any of these allergens (Fig. 1F) . Nevertheless, this enzyme was clearly detected when neutrophils from healthy subjects were treated with LPS (Fig. 1F) , a potent inducer of COX-2 expression in these cells as described [4 ]. However, when neutrophils from allergic patients were cultured with anti-IgG antibodies, no COX-2 expression was detected, as illustrated in Figure 1G for a D1-allergic donor. Furthermore, in no case was COX-2 expression found when neutrophils from allergic donors were treated with nonspecific goat IgG antibodies (Fig. 1 , A, B, and G). Further analysis using RT-PCR indicated that induction of COX-2 mRNA expression took place within 0.5 h of treatment and was maintained for 24 h in neutrophils from allergic patients after challenge with anti-IgE antibodies (Fig. 1H) . In contrast, undetectable levels of COX-2 mRNA were found when these cells were treated with nonspecific goat IgG antibodies.


Figure 1
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  		Figure 1. Induction of COX-2 protein and mRNA expression by antigens and anti-IgE antibodies in human neutrophils, which (A and B) were from an allergic patient sensitized to G3 and cultured in the presence of the indicated concentrations of this antigen or anti-IgE antibodies ({alpha}-IgE) or of 10 µg/ml IgG for 24 h. (C and D) Neutrophils from an allergic patient sensitized to G3 were cultured in the presence of this antigen or anti-IgE antibodies at 10 µg/ml, LPS at 1 µg/ml, or IgG at 10 µg/ml for the times indicated, up to 24 h. Neutrophils from a patient sensitized to M6, T9, and G3 (E) or from a healthy subject (F) were challenged with 10 µg/ml M6, T9, G3, E1, D1, or D2 antigen or with 1 µg/ml LPS for 24 h. (G) Neutrophils from a patient allergic to D1 were cultured for 24 h with anti-IgG ({alpha}-IgG) at the indicated doses or with 1 µg/ml LPS or IgG for 24 h. In all cases, COX-2 protein levels were analyzed by Western blot analysis as described in Materials and Methods. (H) Neutrophils from an allergic patient sensitized to T9 were left untreated or treated with 10 µg/ml anti-IgE antibodies or IgG for the indicated times, and then, COX-2 mRNA levels were analyzed by RT-PCR using COX-2- and GADPH-specific primers. All the experiments were performed at least in triplicate with similar results.

 
Antigen-promoted release of PGE2 and TXA2
To assess whether specific antigens were able to induce PG release, neutrophils from patients sensitized to G3 were cultured with different doses of this antigen or of anti-IgE antibodies for 24 h, and then, PGE2 and TXA2 levels were measured in cell culture supernatants. When unstimulated cells were exposed to the antigen, PGE2 (Fig. 2A ) and TXA2 (Fig. 2B) production was increased by six- to eightfold upon basal levels. This increase was G3 dose-dependent, and a maximal release of PGE2 and TXA2 was obtained at a 10-µg/ml concentration of G3 or anti-IgE antibodies at 24 h of treatment. As also shown, the COX-2 inducer LPS elicited a PGE2 and TXA2 release by neutrophils similar to that induced by anti-IgE antibodies (Fig. 2A and 2B) . Equivalent responses were obtained for other antigens, such as D1 or E1, in patients specifically sensitized to these antigens (data not shown).


Figure 2
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  		Figure 2. Antigen- and anti-IgE antibody-promoted PGE2 and TXA2 release. (A and B) Neutrophils were incubated for 24 h with different doses of the specific antigen (G3) to which the allergic donor was sensitized, with anti-IgE antibody ({alpha}-IgE) or with 1 µg/ml LPS, and the levels of PGE2 (A) or TXA2 (B) were measured in the culture medium supernatants using an enzyme immunoassay. As TXA2 has a short half-life and spontaneously hydrolyzes to TXB2, TXA2 was measured as TXB2. The values shown are the mean ± SE from three independent assays, in which each measurement was performed in triplicate.

 
Role of NADPH oxidase in the control of IgE-dependent COX-2 expression
ROS are recognized as second messengers modulating the expression of several genes in a variety of immune-system cell types [7 , 15 , 20 ]. To evaluate the possible implication of ROS, mainly derived from neutrophil NADPH oxidase activity, in the IgE-dependent COX-2 expression, we first assessed the possibility of IgE-elicited activation of the NADPH oxidase complex taking place upon translocation of its cytosolic subunits, p47phox and p67phox, to the plasma membrane. As shown in Figure 3A , the anti-IgE antibody induced a clear translocation to the plasma membrane of p47phox and p67phox, and there was a concomitant disappearance of these proteins from the cytosolic fraction. Second, as shown in Figure 3B , we found that after treatment with anti-IgE antibody or specific antigens, the enzyme complex was functionally active in the production of ROS and that its activation was cancelled by specific NADPH oxidase inhibitors, such as HMAP, which competes with NADPH for a binding site on the oxidase [23 ], and AEBSF, which blocks the assembly of the NADPH oxidase subunits at the plasma membrane [24 ]. PMA was used in these experiments as a positive control for p47phox and p67phox activation and for ROS generation [8 , 22 ], with consistent results (Fig. 3A and 3B) . To further confirm the implication of ROS arising from the NADPH oxidase system, we analyzed the effect of HMAP and AEBSF on COX-2 expression. As shown in Figure 3C , these two inhibitors completely cancelled the IgE-dependent COX-2 protein synthesis.


Figure 3
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  		Figure 3. Involvement of NADPH oxidase in IgE-dependent COX-2 expression. (A) Neutrophils from an allergic patient sensitized to T9 were treated with 50 nM PMA for 10 min or with 10 µg/ml anti-IgE antibody ({alpha}-IgE) for the times indicated. Then, membrane (left panel) and cytosolic (right panel) fractions were isolated as described in Materials and Methods, resolved by SDS-PAGE, and electrotransferred to PVDF membranes. These were probed sequentially with specific antibodies against the p47 and after stripping p67 subunits of NADPH oxidase. Equal amounts of protein were loaded per lane. Histograms below each lane represent the mean ± SE values quantitated from the blots from three separate experiments. (B) Neutrophils from an allergic patient sensitized to G3 were treated or not with 500 µM HMAP or AEBSF prior to the addition of 50 nM PMA, 10 µg/ml anti-IgE antibody, or 10 µg/ml G3 for 10 min. Then, O2– production was measured by luminol ECL. The values shown are the mean ± SE from three independent assays in which each measurement was performed in triplicate. (C) Neutrophils from an allergic patient sensitized to G3 were left untreated or cultured with 500 µM HMAP or AEBSF for 30 min prior to the addition of 10 µg/ml anti-IgE antibody. After 24 h of treatment, COX-2 expression was analyzed by Western blotting. All the experiments were performed at least in triplicate.

 
H2O2 and specific ROS derived from Fenton reactions are implicated in the COX-2 expression
After passive diffusion through the plasma membrane, H2O2 can be converted into other ROS, such as the oxygen radicals O2·– and ·OH. The most likely mode of intracellular ·OH radical production is via Fenton chemistry, which involves the reduction of H2O2 by ferrous or cuprous [25 ] ions according to the reaction H2O2 + Fe2+/Cu2+ -> ·OH + OH– + Fe3+/Cu3+. Figure 4A shows that exogenously added FeSO4/CuSO4 elicited a strong enhancement of the IgE-dependent COX-2 induction, although without effect by themselves, and that the Fe2+ and Cu2+ chelator PHE inhibited IgE-promoted COX-2 induction. Taken together, these data show an involvement of NADPH oxidase-derived ROS, possibly with ·OH acting as a mediator, in IgE-dependent COX-2 expression.


Figure 4
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  		Figure 4. Effect of H2O2 on COX-2 expression. Involvement of Fenton chemistry. (A) Neutrophils from an allergic patient sensitized to T9 were left untreated or cultured with 20 µM FeSO4 plus 20 µM CuSO4 (Fe2+/Cu2+) or with 20 µM PHE for 30 min prior to the addition of 5 µg/ml anti-IgE antibody ({alpha}-IgE) for 24 h. Then, COX-2 protein expression was analyzed by Western blotting. (B) Neutrophils from an allergic patient sensitized to T9 were cultured in the presence or absence of 25 mM aminotriazole (AMT) and H2O2 at the indicated doses or of 1 µg/ml LPS for 24 h, and COX-2 expression was then analyzed by Western blotting. (C) Neutrophils from an allergic patient sensitized to T9 were cultured with 1 µg/ml LPS or with 25 mM AMT in the absence or presence of 5 µM H2O2 for 5 h, and the levels of COX-2 mRNA were analyzed by RT-PCR. (D) Neutrophils from an allergic patient were preincubated with 25 mM AMT for 30 min. Then, 20 µM FeSO4 plus 20 µM CuSO4 (Fe2+/Cu2+) or 20 µM PHE was added to the culture for 30 min prior to the addition of LPS at 1 µg/ml or of H2O2 at 0.5 or 5 mM, as indicated, and COX-2 expression was analyzed by Western blotting. The results illustrated in each panel are representative of a total of three separate experiments.

 
To further confirm the positive, modulatory role of ROS on COX-2 expression, we analyzed the effect of H2O2 on this process. This compound was found to induce by itself COX-2 expression in a dose-dependent manner at the protein (Fig. 4B) and mRNA levels (Fig. 4C) . Detection of this H2O2 stimulatory effect required, however, the treatment of cells simultaneously with AMT (Fig. 4B and 4C) , an inhibitor of catalase, a H2O2-removing enzyme [26 ]. Next, we assessed the possibility of chemical species subsequently generated from H2O2 through Fenton reactions acting as modulators of COX-2 protein expression. As shown in Figure 4D , the presence of Fe2+/Cu2+ ions notably enhanced the H2O2-induced COX-2 expression. In contrast, the Fe2+ and Cu2+ chelator PHE completely cancelled COX-2 protein accumulation elicited by H2O2 (Fig. 4D) .

Role of MAPK in the control of IgE-dependent COX-2 expression
MAPK family proteins, such as p38, ERK1/2, and JNK1/2, have been implicated in the regulation of COX-2 expression in a broad spectrum of cells [7 , 27 ]. As shown in Figure 5 , neutrophils from an allergic donor sensitized to D1 showed a clear phosphorylation and hence, activation of p38MAPK and ERK1/2 in a time-dependent manner when cultured in the presence of anti-IgE antibodies (Fig. 5A) or of the specific antigen D1 (Fig. 5B) . No activation of these MAPKs was observed, however, in neutrophils from healthy donors when cultured in the presence of a set of antigens or in neutrophils from allergic donors cultured with antigens to which the particular donor was not sensitized (data not shown). In contrast to the results obtained for p38MAPK and ERK1/2, the JNK activation status did not change upon antigen treatment of human neutrophils (data not shown). To study the implication of these signaling pathways on the modulation of COX-2 expression, we tested the effect on this process of PD098059, a specific inhibitor of MAPK kinase [28 ], and of SB203580, a specific inhibitor of p38MAPK [29 ]. Both compounds were found to effectively block anti-IgE antibody-promoted induction of COX-2 expression in neutrophils from allergic donors, whereas IgG had no such effect (Fig. 5C and 5D) .


Figure 5
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  		Figure 5. Involvement of MAPKs in antigen/anti-IgE antibody-dependent COX-2 protein expression. Neutrophils from an allergic patient sensitized to D1 were cultured with 10 µg/ml anti-IgE antibodies ({alpha}-IgE; A) or with 10 µg/ml D1 antigen (B) for the times indicated, and phosphorylated p38MAPK (p38MAPK-P) and ERK1/2 (ERK1/2-P) were subsequently detected. Total (phosphorylated plus unphosphorylated forms) p38MAPK and ERK1/2 are also shown. Neutrophils from an allergic patient sensitized to D1 were preincubated for 45 min with PD098059 (PD; C) or with SB203580 (SB; D) at the doses indicated and then, treated with 10 µg/ml anti-IgE antibody for 24 h. The expression of COX-2 was then analyzed by Western blotting. The results illustrated in each panels are representative of a total of three separate experiments.

 
MAPK pathway is associated with IgE-dependent ROS generation
Given the relationship between the activity of MAPKs and the intracellular redox status in neutrophils [8 , 9 ], we investigated whether such interconnection was operative in the IgE-promoted COX-2 up-regulation. The NADPH oxidase inhibitors, HMAP and AEBSF, were found to completely cancel the IgE-dependent phosphorylation and hence, activation of p38MAPK (Fig. 6A ) and ERK1/2 (Fig. 6B) . As ROS derived from Fenton reactions appeared involved in the IgE-dependent modulation of COX-2 expression, we studied whether these species did contribute to p38MAPK and ERK1/2 activation. As shown, the addition of Fe2+/Cu2+ ions strongly enhanced the IgE-dependent phosphorylation of p38MAPK (Fig. 6C) and ERK1/2 (Fig. 6D) . Conversely, the chelator PHE completely cancelled the anti-IgE antibody-elicited, positive effect on the activation of these two MAPKs (Fig. 6C and 6D) . It is noticeable that there was a robust phosphorylation of ERK1/2 in the presence of Fe2+/Cu2+ alone, i.e., in the absence of anti-IgE antibodies (Fig. 6D) , whereas this was not the case for p38MAPK (Fig. 6C) . These observations are similar to those reported previously in vascular smooth muscle cells [30 ] and fibroblasts [31 ] incubated with different stimuli. These and present observations suggest that ERK1/2 proteins are more sensitive than p38MAPK to small intracellular redox changes in certain cell types. In agreement with these observations, nonlethal concentrations of H2O2 have been demonstrated to activate p38MAPK and ERK1/2 [32 ].


Figure 6
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  		Figure 6. Effect of NADPH oxidase inhibitors and Fenton-promoted cations on MAPKs triggering. Neutrophils from an allergic patient sensitized to T9 were cultured with 500 µM HMAP or AEBSF (A and B), with 20 µM FeSO4 plus 20 µM CuSO4 (Fe2+/Cu2+) or with 20 µM PHE (C and D) for 30 min prior to the addition of 10 µg/ml anti-IgE antibodies ({alpha}-IgE). After a further 30 min of incubation, the cells were lysed, and proteins were analyzed by Western blotting using specific antibodies against the phosphorylated forms of p38 (p38MAPK-P; A and C), ERK1/2 (ERK1/2-P; B and D), or the total phosphorylated plus unphosphorylated forms of these proteins. Results illustrated in each panel are representative of a total of three separate experiments.

 
Involvement of transcription factor NF-{kappa}B on COX-2 up-regulation by anti-IgE antibodies
The involvement of NF-{kappa}B in the activation of COX-2 gene expression [10 ] and its role in inflammatory disorders [12 ] are known. Thus, we next studied the implication of this transcription factor in the IgE-promoted COX-2 expression by using nuclear extracts prepared from anti-IgE antibody- or D1 antigen-treated neutrophils isolated from allergic donors sensitized to this antigen. We observed that the stimulation of these cells with anti-IgE antibody (Fig. 7A ) or D1 antigen (Fig. 7B) resulted in a weak activation of NF-{kappa}B after 30 min of treatment, which was maximal after 2–4 h of treatment and was decreased significantly at 24 h. These data on NF-{kappa}B activation were consistent with previous results from Pouliot et al. [33 ]. Furthermore, the activation by anti-IgE antibody occurred in a dose-dependent manner (Fig. 7C) . The specificity of this DNA-binding activity was evidenced by competition with a 100-fold molar excess of an unlabeled NF-{kappa}B probe (Fig. 7A 7B 7C) . As in unstimulated cells, NF-{kappa}B is anchored to the cytoplasm by a number of inhibitory proteins of the I-{kappa}B family, which become degraded at the proteasome upon cell stimulation with concomitant translocation of NF-{kappa}B to the nucleus [34 , 35 ], we next analyzed the cytoplasmic levels of I-{kappa}B before and after anti-IgE antibody treatment. As shown in Figure 7D , anti-IgE antibody promoted, in a period of 1–4 h, an almost complete degradation of cytosolic I-{kappa}B in human neutrophils, which correlated well in a timely manner with the enhancement of NF-{kappa}B DNA-binding activity (Fig. 7A) . Furthermore, the use of MG-132, a proteasome inhibitor [34 ], strongly attenuated anti-IgE antibody-induced COX-2 protein expression (Fig. 7E) .


Figure 7
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  		Figure 7. Role of NF-{kappa}B in IgE-dependent COX-2 induction. Neutrophils from a patient sensitized to D1 were treated with 10 µg/ml anti-IgE antibody ({alpha}-IgE; A) or 10 µg/ml D1 antigen (B) for the indicated times or with anti-IgE antibody at the indicated doses for 4 h (C). Then, nuclear extracts were prepared, and NF-{kappa}B DNA-binding activity was analyzed by EMSAs. Brackets show the retarded band. (D) Neutrophils from an allergic patient were treated with 10 µg/ml anti-IgE antibody for the indicated times, and I-{kappa}B-{alpha} levels were analyzed by Western blotting in cytosolic extracts. (E) COX-2 expression was analyzed by Western blotting in neutrophils from an allergic patient sensitized to D1, pretreated or not with MG-132 for 2 h at the indicated doses, and then treated with 10 µg/ml anti-IgE antibody for 24 h. (F) Neutrophils from an allergic patient sensitized to T9 were pretreated or not for 1 h with 1 µM SB203580 or 10 µM PD098059 prior to the addition of 10 µg/ml anti-IgE antibody. After 4 h of incubation, nuclear extracts were analyzed for NF-{kappa}B DNA-binding activity. Binding reactions marked with an asterisk (A–C, F) were performed in the presence of a 100-fold molar excess of unlabeled oligonucleotide probe on neutrophils treated with 10 µg/ml anti-IgE antibody for 4 h. Two additional experiments were performed with results similar to those shown in each panel.

 
It has been demonstrated that phosphorylation of the MAPKs p38 and ERK1/2 is a necessary step for NF-{kappa}B activation in some cell types [36 ]. In this context, the presence of SB203580 or PD098059, specific inhibitors of p38MAPK and ERK1/2, respectively, completely inhibited the IgE-dependent NF-{kappa}B DNA-binding activity in human neutrophils (Fig. 7F) . As well, a previous report has shown a positive correlation between H2O2-dependent COX-2 expression and NF-{kappa}B activation in cardiomyocytes [37 ]. We observed in this study that exogenously added H2O2, in the presence of the catalase inhibitor AMT, did enhance NF-{kappa}B DNA-binding activity in neutrophils (Fig. 8A ). Finally, the potential participation of NADPH oxidase on the IgE-dependent NF-{kappa}B activation was also assessed. We found that the two specific NADPH oxidase inhibitors, HMAP and AEBSF, partially cancelled IgE-dependent NF-{kappa}B activation (Fig. 8B) . Strengthening these observations, the presence of Fe2+/Cu2+ ions resulted in an enhancement of the DNA-binding activity of NF-{kappa}B, a process that was cancelled completely by the presence of PHE (Fig. 8B) .


Figure 8
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  		Figure 8. Effect of H2O2 and Fenton-derived ROS on IgE-dependent NF-{kappa}B activation. (A) Neutrophils from an allergic patient sensitized to D1 were treated with 25 mM AMT for 30 min prior to the addition of H2O2 at the indicated doses after 4 h of incubation NF-{kappa}B. DNA-binding activity was analyzed in nuclear extracts. (B) Neutrophils from an allergic patient sensitized to D1 were cultured with or without 500 µM HMAP or AEBSF, 20 µM FeSO4 plus CuSO4 (Fe2+/Cu2+), or 20 µM PHE for 30 min prior to the addition of 5 µg/ml anti-IgE antibodies. After 4 h of incubation, nuclear extracts were prepared, and NF-{kappa}B DNA-binding activity was analyzed. Brackets show the retarded band. Binding reactions marked by an asterisk were performed in the presence of a 100-fold molar excess of unlabeled oligonucleotide in neutrophils treated with 25 mM AMT plus 10 µM H2O2 (A) or with 5 µg/ml anti-IgE antibody (B) for 4 h. Two additional experiments were performed with results similar to those shown.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although the capacity of human neutrophils to induce COX-2 expression in response to inflammatory agonists has been shown [4 ], no studies have been performed yet about the modulation of COX-2 expression by an IgE-dependent mechanism in neutrophils or the molecular mechanisms implicated in this process. The present study unravels that COX-2 expression becomes up-regulated in human neutrophils at the mRNA and protein levels, with a concomitant PGE2 and TXA2 release, in response to challenge with specific antigens or anti-IgE antibodies. This phenomenon raises further questions about the contribution of these cells to inflammatory processes, such as allergy. In this context, the relationship between PGE2 and allergy remains to be well-defined and is currently the subject of different interpretations. Given that increased levels of PGE2 and COX-2 expression have been found in asthmatic subjects [38 ], this finding, together with present data, could point out a role for COX-2 as inducer of the allergy state, and PGs could set to exacerbate the injury on surrounding tissues. In this line, a relationship has been described between COX-2 expression and NADPH oxidase activation and an attenuation for COX-2 inhibition in microglial cells [39 ]. Recently, it has also been evidenced that the PGE2-E-prostanoid receptor 2 (EP2) system contributes to the local production of granulocyte colony-stimulating factor during acute inflammation in mouse peritoneal neutrophils [40 ]. However, another potential interpretation for the association between PGE2 and COX-2 is that COX-2 induction by antigens constitutes a physiological defense mechanism aimed at counteracting the effect of other proinflammatory molecules. In support of the role of PGE2 as a protective molecule in allergic processes, it has been shown that COX-2 inhibition enhances the T helper cell type 2 immune response (e.g., the allergic response) to cutaneous sensitization [41 ]. Also, it has been shown that PGs (e.g., PGD2, PGE2, and PGF2-{alpha}) constitute potent inhibitors of O2·– production by human neutrophils [42 ]. However, recent data have brought into play new, potential signaling molecules, such as PGE2 receptors. In this context, recent studies have highlighted that mice lacking PGE2-EP receptors (EP3 subtype) develop a far more pronounced allergic inflammation than do wild-type mice or mice deficient in other PGE2 receptor subtypes [43 ]. Thus, the PGE2 actions elicited through EP3 receptors could be considered protective against allergic reactions. However, increased numbers of other EP receptors of the EP2 subtype can enhance the sensitivity of asthmatic airway smooth muscle cells to PGE2 [44 ]. Therefore, the balance of EP3/EP2 receptor expression has been postulated as a key factor in the development of the allergic state. In contrast, a positive relationship between TXA2 and allergy seems well-accepted [45 ].

It is important to note that the three known forms of IgE receptors, namely Fc receptor for IgE (Fc{epsilon}R)I, Fc{epsilon}RII/CD23, and galectin-3 [46 ], are present in neutrophils, thus supporting the potential participation of these cells in allergic processes [13 14 15 ]. In this regard, we have shown that specific antigens are able to activate several functional responses by neutrophils from allergic patients and highlighted the presence of specific IgE molecules bound to antigens on the surface of neutrophils [15 , 16 ]. As well, we have reported the absence of IgG molecules specific for the antigens to which the patients were sensitized [15 ]. We have thus postulated that the mechanisms whereby antigens promote neutrophil activation could well be related to the binding of antigens to specific IgE molecules associated with their specific receptors at the neutrophil plasma membrane. This mechanism would explain why functional responses elicited by antigens, such as induction of COX-2 expression, are not observed in neutrophils from healthy subjects or from allergic patients treated with antigens other than those to which they had previously become sensitized. In the latter case, antigens would not bind to pre-existent IgE/receptor complexes, and ensuing COX-2 induction would not take place.

It is well known that stimulation of phagocytic cells induces a set of phenomena, known collectively as the respiratory burst, characterized by an increase in the production of O2·– anions [16 , 47 ]. The major O2·–-generating enzyme in neutrophils is NADPH oxidase. Upon its reduction, O2 may sequentially generate O2·–, which is rapidly converted into H2O2 by the action of superoxide dismutase [48 ], and ·OH in the presence of Fe2+/Cu2+ cations, through the so-called Fenton reaction. Previously, we have demonstrated that in vitro challenge of human neutrophils with antigens to which the patients were sensitized or with anti-IgE antibody elicited a greater release of ROS than that found in neutrophils from healthy subjects [16 ]. Recent studies have highlighted a connection between this up-regulation of COX-2 expression and the generation of ROS during monocyte-to-macrophage differentiation [7 ]. It is interesting that neutrophils from allergic subjects have been shown to generate higher levels of O2·– than those isolated from healthy individuals [49 ]. However, the molecular mechanisms mediating COX-2 gene induction in a wide range of cells, including neutrophils, remain unclear at present.

In agreement with our previous report [16 ], we now describe that specific antigens/anti-IgE antibodies were able to elicit a clear activation of the NADPH oxidase complex in neutrophils, concomitant with the translocation to the plasma membrane of its cytosolic p47phox and p67phox subunits and the subsequent release of ROS. This process was abrogated by HMAP and AEBSF, two specific NADPH oxidase inhibitors, which were also found to cancel antigen/anti-IgE antibody-dependent COX-2 expression. Moreover, as the addition of Fe2+/Cu2+ cations or of the Fe2+/Cu2+ chelator PHE enhanced or inhibited, respectively, the induction of COX-2 expression promoted by antigens or anti-IgE antibodies, oxygen species derived from Fenton reactions could constitute the causal effectors of COX-2 up-regulation.

Taken together, present data suggest that the induction of COX-2 expression promoted by specific antigens/anti-IgE antibodies is modulated by ROS, possibly acting through the activation of MAPK-signaling pathways and the transcription factor NF-{kappa}B. In this context, the interplay among MAPK-dependent pathways, NF-{kappa}B activation, and ROS is largely known in neutrophils [8 , 9 ]. In this light, ERK1/2 and p38MAPK have been implicated previously in the induction of COX-2 expression in monocytes [7 ]. The complexity of the interactions operative among these pathways was evidenced by the fact that NADPH oxidase inhibitors cancelled the activation of p38MAPK and ERK1/2 promoted by anti-IgE antibody treatment and that SB203580 and PD098059, two inhibitors of these MAPKs, provoked a strong down-regulation of COX-2 expression. Therefore, present data underscore that the positive action of ROS production on IgE-dependent COX-2 induction is likely exerted via the activation of the MAPKs p38 and ERK1/2.

In addition, the relevance of NF-{kappa}B-binding sites in the COX-2 promoter and their role in development of the allergic state are well-established [11 , 12 ]. In the present work, we show evidence that the stimulation of neutrophils with anti-IgE antibodies or D1 antigen resulted in a weak activation of NF-{kappa}B in a matter of minutes, followed by maximal activation after 2–4 h of treatment. These data on NF-{kappa}B activation were consistent with previous results from Pouliot et al. [33 ]. Also, we have observed that degradation of cytoplasmic I-{kappa}B becomes triggered upon anti-IgE antibody treatment with concomitant activation of NF-{kappa}B in the nucleus. The relationships between NF-{kappa}B activation and the referred cytosolic signaling pathways are evidenced further by the fact that specific inhibitors of MAPKs or of NADPH oxidase also negatively affected NF-{kappa}B DNA-binding activity. In summary, present observations support a role for intracellular oxidants as participants in a novel molecular mechanism, elicited by antigens or anti-IgE antibodies, leading to COX-2 induction in human neutrophils during the development of the allergic state. Our data establish the existence of intracellular signal transduction mechanisms, such as O2·– release, MAPK, and NF-{kappa}B activation, although the exact hierarchical order of these pathways needs to be clarified still.


   ACKNOWLEDGEMENTS
 
A. V. and P. C. were supported by fellowships from the Ministerio de Educación y Ciencia and G. A., by the Junta de Andalucía (J.A.), Spain. This work was funded by Grant SAS-74/04 from the Consejería de Salud, J.A., awarded to F. S. A. V. and P. C. contributed equally to this work.

Received July 25, 2005; revised February 22, 2006; accepted February 24, 2006.


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