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The Journal of Immunology, 2007, 179, 2634-2641
Copyright © 2007 by The American Association of Immunologists, Inc.
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Neutrophils as a Novel Source of Eosinophil Cationic Protein in IgE-Mediated Processes1

Javier Monteseirín2,3,*, Antonio Vega3,*, Pedro Chacón3,*, M. Jesús Camacho*, Rajaa El Bekay{dagger}, Juan A. Asturias{ddagger}, Alberto Martínez{ddagger}, Pedro Guardia*, Ramón Pérez-Cano§ and José Conde*

* Servicio Regional de Inmunología y Alergia, Hospital Universitario Virgen Macarena, Sevilla, Spain; {dagger} Fundación IMABIS, Laboratorio de Investigación, Hospital Civil, Málaga, Spain; {ddagger} Bial-Aristegui, Research and Development Department, Bilbao, Spain; and § Departamento de Medicina, Universidad de Sevilla, Sevilla, Spain


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The production of eosinophil cationic protein (ECP) in IgE-mediated diseases has been associated mainly with eosinophils, although no IgE-dependent ECP release has been observed in these cells. Because there is increasing evidence of neutrophil participation in allergic processes, we have examined whether human neutrophils from allergic patients were able to produce ECP by an IgE-dependent mechanism. After challenge with specific Ags to which the patients were sensitized, ECP release was detected in the culture medium. Furthermore, intracellular protein was detected by flow cytometry, immunofluorescence staining, and Western blotting. Expression at both mRNA and de novo protein synthesis were detected, respectively, by RT-PCR and radiolabeling with 35S. Ag effect was mimicked by cell treatment with anti-IgE Abs or Abs against Fc{epsilon}RI and galectin-3 (Fc{epsilon}RI>galectin-3), but not against Fc{epsilon}RII. These observations represent a novel view of neutrophils as possible source of ECP in IgE-dependent diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Eosinophil cationic protein (ECP)4 is a highly cationic protein, belonging to the RNase superfamily, with potent cytotoxic properties against mammalian and nonmammalian cells such as parasites, bacteria, and viruses. In addition to being cytotoxic, ECP has a number of noncytotoxic activities, including regulation of immune and epithelial cells, complement, coagulation, and fibrinolysis (1, 2, 3). Despite this strong association between eosinophils and allergy, the mechanisms of cellular activation are largely unknown. Expression of IgE receptors on eosinophils had been reported in cells from patients with marked eosinophilia (4, 5, 6, 7), but not in cells from healthy donors and/or subjects with mild-to-moderate eosinophilia. In addition, IgE-dependent functional responses, such as ECP release, were not observed in blood eosinophils from healthy and allergic individuals, whereas these eosinophils responded vigorously to IgG (8, 9, 10). mAbs against ECP have been used to detect total eosinophils. Immunostaining techniques evidenced that the number of ECP+ cells was higher than the number of eosinophils. It has been shown that neutrophils also contain ECP (11, 12, 13, 14, 15, 16, 17, 18). Furthermore, the ECP was specifically associated with neutrophils in allergic-asthmatic processes (19, 20, 21, 22, 23, 24). There is increasing evidence of neutrophil participation in asthma and the allergic process (25). For instance, peripheral blood neutrophils are activated during active asthma (26), after exercise-induced bronchospasm (27), and during both early and late allergen-induced asthmatic reactions (28). Several studies have reported evidence linking the presence of neutrophils with airway damage and dysfunction (29, 30, 31). IgE can bind on neutrophils at the high-affinity receptor for IgE (Fc{epsilon}RI) (32), the low-affinity receptor for IgE (Fc{epsilon}RII/CD23) (33, 34), and galectin-3 (35, 36). We have previously shown that specific Ags were able to activate functional responses by neutrophils from allergic patients sensitized to Ags of the same type as those which produce clinical allergic symptoms. For example, allergens induce in neutrophils the release of myeloperoxidase, IL-8, and elastase, the down-modulation of L-selectin, and the respiratory burst and intracellular increase of Ca2+ levels (37, 38, 39, 40, 41). If eosinophils are unable to release ECP by an IgE-dependent mechanism, can neutrophils do so? The present work was undertaken to analyze this question.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Chemicals and reagents

The Ags were commercially available Ag extracts, including D1 (Dermatophagoides pteronyssinus), G3 (Dactylis glomerata), T9 (Olea europaea), and W6 (Artemisia vulgaris). They were purchased from Bial-Aristegui. Goat anti-human IgE Ab, goat anti-human IgG Ab, and the Fix-and-Perm cell kit were obtained from Caltag Laboratories. Goat or mouse IgG, L-{alpha}-phosphatidylcholine, platelet-activating factor (PAF) (beta-acetyl-{gamma}-O-alkyl), and LPS were from Sigma-Aldrich. Ficoll-Hypaque, PBS, RPMI 1640, FBS, and penicillin/streptomycin were purchased from BioWhittaker. PCR primers and protein G-Sepharose were obtained from Amersham Pharmacia Biotech. Anti-human CD9 Ab, anti-human CD16 Ab, anti-human CD203c Ab, anti-human CD66b Ab, anti-human Fc{epsilon}RII/CD23 Ab (clone 9P.25), FITC-conjugated goat anti-mouse IgG Ab, and goat anti-mouse IgG micromagnetic beads were obtained from Beckman Coulter-IZASA. Polyvinylidene difluoride (PVDF) membranes were from Bio-Rad. mAb against human ECP was from Pharmacia Diagnostics. Horseradish-conjugated anti-mouse IgG Ab was obtained from Promega. Anti-human galectin-3 Ab (clone A3A12) and anti-human Fc{epsilon}RI {alpha}-chain Ab (clone AER-37/CRA1) were purchased from Abcan and eBioscience, respectively. 35Izolabel (35S) was obtained from Nucliber. Kodak X-Omat AR films were obtained from Kodak. Mouse mAb specific for human anti-beta-actin (sc-8432) was purchased from Santa Cruz Biotechnology. All cultured reagents had endotoxin levels of ≤0.01 ng/ml, as tested by the Limulus lysate assay (Coatest; Chromogenix).

Patients and controls

The studied group included adult atopic patients with intermittent bronchial asthma and healthy adult nonatopic volunteer controls (42). The group of asthmatic patients had positive skin-prick test (Bial-Arístegui) and specific IgE (HYTEC 288; Hycor Biomedical-IZASA) to at least one common Ag. The subjects received no specific hyposensitization. The patients were not allowed to take any bronchodilators within the 8 h before challenge of cells in vitro. Oral bronchodilators were withheld for 24 h, and none of the subjects had taken corticosteroids, disodium cromoglycate, or nedocromil sodium in the previous week. The healthy controls had no history of allergy or bronchial symptoms, and had negative skin-prick test (Bial-Arístegui) and specific IgE (HYTEC 288) to a battery of inhalant Ags (house-dust mites, pollens, molds, and animal danders). The hospital ethics committee approved the study and each subject gave informed consent.

Preparation of neutrophils and eosinophils

Human neutrophils were isolated as previously described (37, 38, 39, 40, 41). Neutrophil preparations were further purified using a MACS by incubation with mouse anti-human CD9, anti-human CD203c, and anti-human CD14 Abs, and then with anti-mouse IgG micromagnetic beads. The purity of neutrophils was on average >99%. To prepare mRNA for RT-PCR, neutrophils were purified even further by repeating the procedure above thrice more, using anti-CD9, anti-human CD203c, and anti-human CD14 Abs each time, which reduced contaminating eosinophils to 0.001–0.004% of the final cell population. Human eosinophils were analogously purified by MACS, using anti-CD16, anti-CD203, and anti-human CD14 Abs as previously described (43).

Neutrophil and eosinophil culture: treatment with different agents

Neutrophils and eosinophils were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 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. For stimulation treatments, cells were incubated with Ags, anti-human IgE Ab, goat IgG, or agonists at 37°C for the times indicated in figure legends. LPS was used at 1 µg/ml (44). None of the reagents affected the viability of the cells at the concentrations used in this work, as confirmed by the trypan blue dye-exclusion test.

ECP release

ECP released was measured in the culture supernatants by ELISA (CAP system immunoassay; Pharmacia) according to the manufacturer’s instructions.

ECP expression analysis by flow cytometry

Neutrophils (2 x 106, cultured at a density of 6.6 x 106/ml) were cooled on ice and washed with cold PBS. Cells were incubated with PE-conjugated anti-CD66b Ab or the IgG1-PE isotype control Ab, for 15 min. After washing with PBS, cells were fixed and permeabilized using the Fix-and-Perm cell kit, following the manufacturer’s indications. Next, cells were incubated for 20 min with anti-human ECP (1 µg), washed with PBS, and pelleted by centrifugation. Then, cells were incubated with FITC-conjugated goat anti-mouse IgG at 4°C in the dark for 30 min, washed, and finally resuspended in PBS. In the isotype control, the primary Ab was omitted. Flow cytometry analysis was performed using an Epics XL-MCL system (Coulter-IZASA). For each sample, 10,000 events were analyzed for fluorescence intensity. Results were expressed as frequency of fluorescence-positive cells (%).

Immunofluorescence staining

After treatment, 5 x 106 cells (cultured at a density of 6.6 x 106/ml) were harvested, washed with PBS, and smeared onto poly-L-lysine-treated glass slides. The slides were fixed at room temperature for 10 min with 2% paraformaldehyde. After washing with PBS, cells were blocked for 15 min with 1% BSA in PBS containing 0.1% Triton X-100, then incubated with primary mouse anti-human ECP Ab (2 µg/ml) for 1 h, washed extensively, and stained for 30 min with secondary FITC-conjugated goat anti-mouse IgG Ab (1:100). After extensive washing, coverslips were mounted on the slides using mounting medium (10% PBS, 90% glycerol). Immunostained cells were observed and photographed using a Nikon EFD-3 microscope (Nikon-IZASA). In negative controls, the primary Ab was not added.

Western blotting analysis

Cells (107, cultured at a density of 6.6 x 106/ml) were pelleted, lysed in 50 µl of ice-cold lysis buffer A (50 mM Tris-HCl (pH 7.9), 10 mM EDTA, 50 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 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 PMSF, 1 mM benzamidine, and 10 mM iodoacetamide), and vortexed for 10 s following sonication for 10 s; insoluble material was pelleted by centrifugation at 15,000 x g for 2 min. The supernatants were used for protein measurement. Proteins (80 µg/lane) were separated on 15% polyacrylamide-SDS gels under reducing conditions, and electrophoretically transferred to PVDF membranes using a semidry device. Membranes were probed without need of prior blocking (45) with anti-human ECP Ab (1:500), at room temperature for 2 h, in PBS containing 1% BSA and 0.2% Tween 20. Next, membranes were washed with PBS, and incubated at room temperature for 30 min with horse anti-mouse IgG Ab linked to HRP (1:5000). After washing with PBS, immunoreactive bands were visualized by ECL (46). Reprobing of blots with anti-human beta-actin Ab was conducted to verify even protein loading throughout lanes.

Dissociation of neutrophil-bound Igs

Ig molecules were dissociated from the surface of neutrophils as described previously (39, 40). Briefly, after isolation, neutrophils were resuspended in 1 ml acetate buffer (50 mM sodium acetate (pH 4), 85 mM NaCl, 5 mM KCl, supplemented with 0.03% human serum albumin) and incubated on ice for 3 min. An equal volume of gelatin veronal buffer (1.8 mM sodium barbital, 3.1 mM barbituric acid, 0.1% gelatin, 0.05 mM MgCl2, 141 mM NaCl, 0.15 mM CaCl2 (pH 7.4)) was then added to the treated cells, and the mixture was centrifuged at 500 x g for 10 min. After the treatment, neutrophils were cultured with the different agents.

RNA extraction and RT-PCR analysis

Total RNA from 2 x 107 highly purified neutrophils or eosinophils (cultured at a density of 6.6 x 106/ml) was isolated using the guanidine phenol method (47). Total RNA (1 µg) was reverse transcribed into cDNA using random primers. The first-strand cDNA was amplified with primer sets for human ECP (accession number: X55990): 5'-GCACATCAGTCTGAACCCCCCTCG-3' and 5'-TAGAACCTCCTTCCTGGTCTGTCT-3', GAPDH (accession number: J04038): 5'-CCACCCATGGCAAATTCCATGGCA-3' and 5'-TCTAGACGGCAGGTCAGGTCCACC-3', Charcot-Leyden crystal protein (CLC) (accession number: NM_001828): 5'-AGGAGACAACAATGTCCCTG-3' and 5'-TCACAGCCTCAGGCTTGATT-3'. The reaction was performed by 40 cycles each of 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min. The PCR products were electrophoresed through agarose gels, and bands were visualized by ethidium bromide staining (ECP: 280 bp, GAPDH: 600 bp, CLC: 373 bp).

De novo synthesis of ECP

Cells (2 x 107, cultured at a density of 6.6 x 106/ml) were cultured in methionine- and cysteine-free medium (RPMI 1640 medium containing 10% (v/v) dialyzed FBS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin) supplemented with 250 µCi/ml [35S]methionine and [35S]cysteine (35Izolabel 35S). Cells were incubated for 18 h, at 37°C, in an atmosphere of 5% CO2 and 95% O2. Following incubation, cells were harvested and centrifuged (12,000 x g for 10 min, at 4°C), and cell culture supernatants were collected and kept at –80°C. Pelleted cells were lysed in 1 ml of cold buffer (50 mM Tris (pH 7.4), 10 mM EGTA, 50 mM NaCl, 1% Triton X-100, 2 mM sodium orthovanadate, 100 µM phenylarsine oxide, and 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 PMSF, 1 mM benzamidine, and 10 mM iodoacetamide). After 10 min, cells were vortexed for 10 s, sonicated for 10 s, and centrifuged at 12,000 x g for 2 min at 4°C. The supernatants obtained after that centrifugation constituted whole cell extracts. Whole cell extracts (1 ml~1 mg) and cell culture supernatants previously obtained (1 ml) were incubated with 2 µg/ml anti-human ECP Ab for 18 h, at 4°C, and then with 75 µl of IgG-coupled protein G-Sepharose for 4 h at 4°C. Next, Sepharose was pelleted, and washed twice with 20 mM sodium phosphate buffer (pH 7.0) containing the previous protease-mixture inhibitor. Immunoprecipitated proteins were subjected to SDS-PAGE by boiling the Sepharose particles in Laemmli sample buffer, on 15% polyacrylamide gels under reducing conditions. Following electrophoresis, the gels were dried, mounted on Kodak X-Omat AR films with an intensifying screen, and exposed for 1–5 days at –80°C.

Statistical analysis

Data are expressed as means ± SEM. A Student t test or one-way ANOVA was used to make comparisons between groups. A level of p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Specific Ags induce release of ECP from human neutrophils, but not from human eosinophils

The potential ability to release ECP was evaluated in highly purified neutrophils and eosinophils from allergic asthmatic patients. For this, the cells were cultured in the presence of Ags to which the patients were sensitized, or of agonists, including PAF and LPS, and the release of ECP was measured in the cell culture supernatant by ELISA. As is shown in Fig. 1A, the treatment with Ags and/or PAF induced a time-dependent ECP release by neutrophils, but not by eosinophils (Fig. 1B). In contrast, LPS induced a time-dependent ECP release only by eosinophils (Fig. 1B).


Figure 1
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  		FIGURE 1. Kinetics of ECP release by human neutrophils and eosinophils. Neutrophils (A) or eosinophils (B) from allergic patients were left untreated or were incubated with Ag to which the patients were sensitized (10 µg/ml) and/or PAF (5 µg/ml), or LPS (1 µg/ml), for the times indicated, and the levels of ECP in the culture medium supernatants were determined using an enzyme immunoassay. The values shown are the mean ± SEM from seven independent assays in which each measurement was performed in duplicate. C, Total RNA was extracted from highly purified unstimulated neutrophils from seven human allergic patients (N1-N7), as well as from highly purified eosinophils (E) used as positive control. RNA samples were analyzed by RT-PCR, using specific primers for the amplification of the CLC transcripts. Results from amplification of the GAPDH housekeeping gene transcript are shown.

 
When neutrophils were cultured with Ags alone, ECP release was significant (p = 0.013) only at 18 h. However, when cells were cultured with Ags together with PAF, ECP release was detected at 3 and 6 h, indicating that PAF acted additively with Ags.

Because it has been reported that eosinophils and basophils (48, 49, 50, 51, 52), but not neutrophils, can express CLC, we additionally analyzed, by RT-PCR, the expression of its transcript, and thereby excluded a possible eosinophil and basophil contamination in our preparations. As is shown in Fig. 1C, no C-L mRNA was detected in any neutrophil sample, whereas CLC expression was found in the eosinophil sample examined. Fig. 2A illustrates a dose-dependent ECP release after challenge of neutrophils for 18 h with different doses of the specific Ags to which the donor was sensitized. As is also shown, the maximal level of ECP release was attained after treatment with 10 µg/ml Ag.


Figure 2
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  		FIGURE 2. Ag dose-dependent ECP release response by human neutrophils. Specificity of Ag response. A, Neutrophils from allergic patients (n = 7) were left unchallenged or were challenged for 18 h with different doses of the specific Ag to which the donors were sensitized. B, Neutrophils obtained from healthy donors, nonsensitized asthmatic patients, or sensitized asthmatic patients were untreated or treated with 10 µg/ml Ag for 18 h. In all cases, levels of ECP in the culture supernatants were determined by ELISA. Data are the mean ± SEM of seven experiments conducted on seven different cell preparations.

 
Next, experiments were performed to study the specificity of the response to Ags. Neutrophils from a group of asthmatic patients, cultured with Ags to which the patients were sensitized, showed a significantly higher ECP production than that of cells from healthy donors, or cells from asthmatic patients, incubated with an Ag to which patients were not sensitized (p < 0.001). No statistical differences were observed between unstimulated cells, cells from healthy donors, and cells from allergic subjects, cultured in the presence of Ags to which the patients were not sensitized (Fig. 2B).

Role of IgE in ECP release by human neutrophils

We have previously reported the ability of neutrophils from allergic patients to bind Ag-specific IgE on their cell surface (39, 40). In this regard, we further investigated the participation of IgE in Ag-induced ECP release. First, we studied whether anti-human IgE Ab treatment could mimic the effect observed with Ags. Fig. 3 shows that anti-IgE Ab, like Ags, induced ECP release in a time- (Fig. 3A) and dose- (Fig. 3B) dependent manner, with a maximal production at 18 h, at 10 µg/ml anti-IgE Ab. An additive effect similar to that with Ags was observed in the presence of PAF (Fig. 3A). In no case was ECP release found when neutrophils from allergic patients were treated with nonspecific goat IgG Ab. In addition, goat IgG did not affect the PAF-dependent ECP release by neutrophils. Furthermore, when neutrophils from allergic patients were cultured with anti-IgG Abs, no ECP release was detected (Fig. 3A). Moreover, ECP release was not detected when IgE molecules were stripped from the neutrophil surface before the Ag challenge (39) (Fig. 3C).


Figure 3
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  		FIGURE 3. Anti-IgE Ab time- and dose-dependent ECP release by human neutrophils. A, Neutrophils from allergic patients were untreated or treated with anti-IgE Ab ({alpha}-IgE; 10 µg/ml), PAF (5 µg/ml), PAF (5 µg/ml) plus anti-IgE (10 µg/ml), goat IgG (IgG; 10 µg/ml), PAF (10 µg/ml) plus goat IgG (10 µg/ml), or anti-IgG ({alpha}-IgG; 10 µg/ml), where indicated for the indicated times. B, Neutrophils from allergic patients were untreated or treated with anti-IgE Ab or goat IgG at the indicated doses for 18 h. C, Cells from an allergic patient were left untreated or were treated to elute the surface Ig and then challenged with an Ag to which the neutrophil donor was sensitized (10 µg/ml), or PAF (5 µg/ml) as a positive control of ECP release, for 18 h. Levels of ECP release in cell culture supernatant were determined by ELISA. Data shown are the mean ± SEM from seven separate experiments in which measurements were performed in duplicate.

 
The presence of the three forms of IgE-receptor/IgE-binding molecule (Fc{epsilon}RI, Fc{epsilon}RII/CD23, and {epsilon}BP/mac-2/galectin-3) has previously been reported in neutrophils (32, 33, 34, 35, 36). The next experiments were performed to identify the types of IgE receptor/IgE-binding molecule involved in IgE-dependent ECP release. Fig. 4A illustrates a higher ECP release by neutrophils from an allergic patient, incubated with CRA1 Ab (directed against the Fc{epsilon}RI {alpha}-chain), compared with neutrophils treated with A3A12 Ab (directed against galectin-3). However, when neutrophils were challenged with 9P.25 Ab (directed against Fc{epsilon}RII/CD23), the amount of ECP released was similar to that in the case of unstimulated cells. In no case was ECP release found when neutrophils from allergic donors were treated with nonspecific mouse IgG1 Ab. In contrast to neutrophils, eosinophils responded only to the treatment with LPS (Fig. 4B). Galectin-3 activity is affected by lactose, which is a saccharide ligand recognized by galectin-3 (53). To further confirm the involvement of this IgE-binding protein in IgE-dependent ECP release by human neutrophils, experiments were next done to clarify whether lactose could inhibit the Ag-dependent ECP release. Fig. 4C shows a partial inhibition of Ag-dependent ECP release by lactose, while preincubation with glucose did not affect the Ag-dependent ECP release.


Figure 4
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  		FIGURE 4. Anti-Fc{epsilon}Rs/anti-galectin-3-elicited ECP release by human neutrophils. Effect of lactose. Neutrophils (A) and eosinophils (B) from allergic patients (n = 7) were incubated, where indicated, with anti-Fc{epsilon}RI Ab ({alpha}-Fc{epsilon}RI; 5 µg/ml), anti-Fc{epsilon}RII/CD23 Ab ({alpha}-CD23; 5 µg/ml), anti-galectin-3 Ab ({alpha}-galectin-3; 5 µg/ml), PAF (5 µg/ml) plus anti-IgE Ab ({alpha}-IgE; 10 µg/ml), PAF (5 µg/ml) plus goat IgG (10 µg/ml), LPS (1 µg/ml), or mouse IgG1 (5 µg/ml) for 18 h. C, Neutrophils from an allergic patient were preincubated with 25 mM lactose or 25 mM glucose before the addition of the Ag to which the patient was sensitized (10 µg/ml) for 18 h. Levels of ECP release in cell culture supernatant were determined by ELISA. Data shown are the mean ± SEM from seven separate experiments in which measurements were performed in duplicate.

 
Intracellular expression of ECP in neutrophils

Additional experiments were performed to analyze whether, in addition to releasing ECP, human neutrophils were able to express ECP protein. To this end, we studied the intracellular ECP expression in human neutrophils. After two-color flow cytometry analysis, we detected that a low number of nonstimulated neutrophils (CD66b+ cells) displayed intracellular ECP expression (Fig. 5A). However, this expression was clearly enhanced after 18 h of incubation with Ag to which the patient was sensitized, anti-IgE Ab, and PAF. As is shown, PAF additively enhanced the effect of Ag and anti-IgE Ab. Further analysis performed using immunofluorescence staining yielded similar results (Fig. 5B). Additional analysis by Western blotting (Fig. 5C) showed that ECP protein was only weakly detected in untreated cells; however, ECP was detected after challenge with anti-IgE Ab, PAF, or PAF plus anti-IgE Ab. In no case was ECP expression found when neutrophils from allergic donors were treated with nonspecific goat IgG Ab or anti-IgG Ab. In addition, goat IgG did not affect the PAF-dependent ECP expression by neutrophils.


Figure 5
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  		FIGURE 5. Analysis of neutrophil intracellular ECP expression. A, Neutrophils isolated from an allergic patient were cultured for 18 h in the absence of stimulus (a) or in the presence of goat IgG (IgG; 10 µg/ml) (b), Ag (10 µg/ml) (c), PAF (5 µg/ml) (d), anti-IgE Ab ({alpha}-IgE; 10 µg/ml) (e), PAF (5 µg/ml) plus anti-IgE Ab (10 µg/ml) (f), PAF (5 µg/ml) plus Ag (10 µg/ml) (g), or PAF (5 µg/ml) plus goat IgG (10 µg/ml) (h) as indicated in the figure. Intracellular ECP and cell surface CD66b expression was subsequently analyzed by flow cytometry. The results from a representative experiment of three with similar findings are shown. The numbers to the right of each plot represent the percent of double-positive cells (CD66b+/ECP+). B, Neutrophils from an allergic patient were processed as indicated in Materials and Methods for light microscopy (left column) or intracellular ECP detection by fluorescence microscopy (right column). Untreated cells (a) and cells after 18 h of treatment with goat IgG control Ab (10 µg/ml) (b), PAF (5 µg/ml) (c), anti-IgE (10 µg/ml) (d), PAF (5 µg/ml) plus anti-IgE Ab (10 µg/ml) (e), or PAF (5 µg/ml) plus goat IgG (10 µg/ml) (f) are shown. Two separate experiments were performed with similar results. C, Neutrophils from an allergic donor were immediately lysed after isolation (basal) or were left untreated or treated, where indicated, with goat IgG (10 µg/ml), anti-IgG Ab (10 µg/ml), PAF (5 µg/ml), anti-IgE (10 µg/ml), PAF (5 µg/ml) plus anti-IgE (10 µg/ml), or PAF (5 µg/ml) plus goat IgG (10 µg/ml) for 18 h. Cells were lysed, and the proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and sequentially probed with anti-human ECP and anti-human beta-actin. Three separate experiments were performed, with similar results.

 
IgE-dependent ECP mRNA expression in neutrophils from allergic patients

Although the presence of ECP protein has previously been found in neutrophils (11, 12, 13, 14, 15, 16, 17, 18), its mRNA expression has never been detected in these cells. Therefore, we next studied the observed effects of anti-IgE Ab, Ags, and PAF upon ECP expression at mRNA levels, using RT-PCR. Because RT-PCR is a very sensitive method, it was important to isolate RNA from extremely pure cells to avoid false-positive results. Neutrophil and eosinophil populations were close to 100% purity (see Materials and Methods). Fig. 6 shows the absence of ECP mRNA in unstimulated neutrophils (Fig. 6A) and its presence in unstimulated eosinophils (Fig. 6B). Bands of amplification product corresponding to ECP were present in neutrophils stimulated with PAF, anti-IgE alone, or anti-IgE Ab together with PAF (Fig. 6A). Unspecific goat IgG was taken as negative control, and did not exert any effect on ECP mRNA levels (Fig. 6A). mRNA amplification of the CLC was also assayed to exclude eosinophil and basophil contamination (48, 49, 50, 51, 52) (Fig. 6C).


Figure 6
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  		FIGURE 6. Analysis of ECP mRNA expression by human neutrophils and eosinophils. Neutrophils (A) and eosinophils (B) from allergic patients were either left without any treatment or treated with goat IgG Ab (IgG; 10 µg/ml), PAF (5 µg/ml), anti-IgE ({alpha}-IgE; 10 µg/ml), or PAF (5 µg/ml) plus anti-IgE (10 µg/ml) for 5 h. After total RNA extraction, RNA samples were analyzed by conventional RT-PCR, using specific primers for the amplification of ECP transcript (A and B). C, RNA samples shown in A were analyzed in parallel with eosinophils (E) for the amplification of the CLC transcripts (C). Results from amplification of the GAPDH housekeeping gene transcript are shown in the three panels. Similar results were obtained in two additional experiments.

 
De novo synthesis of ECP by human neutrophils

For further confirmation, in addition to the presence of ECP in human neutrophils, ECP de novo synthesis was analyzed in these cells. Human neutrophils were cultured with anti-IgE Ab plus PAF for 20 h in a medium containing [35S]methionine and [35S]cysteine. Then, cellular lysates or supernatants were immunoprecipitated using a mAb against ECP. Results presented in Fig. 7 show that treatment of neutrophils with these agents induced de novo synthesis of ECP. In addition, ECP synthesis was detected in both cellular lysates and cell culture supernatants, indicating that ECP release, at least in part, was originated in de novo synthesis of the protein.


Figure 7
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  		FIGURE 7. De novo biosynthesis of ECP by human neutrophils. Human neutrophils from an allergic patient were cultured with PAF (5 µg/ml) plus anti-IgE Ab ({alpha}-IgE; 10 µg/ml) in the presence of a medium with [35S] methionine and cysteine. Subsequently, the supernatants were collected and the cell pellets were lysed. Supernatants and lysates were immunoprecipitated with an Ab against ECP. Proteins were resolved by SDS-PAGE, and the immunoreactive bands were visualized by autoradiography. Similar results were obtained in two additional experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results described here show, for the first time, that the IgE-dependent mechanism is an effective stimulus for synthesis and release of ECP by human neutrophils. Recent studies show that ECP is not only a distinctive eosinophil protein, but has been found in neutrophils (11, 12, 13, 14, 15, 16, 17, 18). In this regard, it has been reported that these cells are able to take up ECP from the bloodstream after phagocytosis and store it, but not to synthesize it (13). In agreement with these reports, our results indicate that in resting (unstimulated) neutrophils, no ECP mRNA was detected, and only a small amount of intracellular or released protein was found. However, after cellular stimulation, ECP was synthesized. This was verified by several lines of evidence: 1) after neutrophil stimulation, bands of PCR product corresponding to ECP mRNA were detected; 2) de novo biosynthesized protein was detected by 35S radiolabeling; 3) an increase of intracellular ECP protein was observed by flow cytometry, fluorescence microscopy, and Western blotting; and 4) an accompanying ECP release was detected by ELISA.

The results described here provide the first evidence of a new mechanism of ECP induction in human neutrophils. This mechanism, elicited by Ags, was highly specific. In this context, Ags other than those which produce clinical symptoms did not evoke ECP production, and were ineffective in healthy donors. Presumably, the Ag-mediated effect occurred in an IgE-dependent manner. This hypothesis is supported by four main facts: 1) we have previously described the presence of specific IgE on the surface of neutrophils from allergic patients (39, 40); 2) anti-IgE Ab challenge triggered an equivalent response to that obtained with the Ags, and this response was not detected after IgE stripping from the cell surface; 3) anti-IgG Ab challenge did not induce ECP expression or release by human neutrophils; and 4) ligation of Fc{epsilon}RI and galectin-3 with specific Ab evoked an ECP release.

In line with our results, IL-8 liberation by these cells following stimulation with anti-Fc{epsilon}RI Ab has been reported previously (32); moreover, galectin-3 stimulates superoxide production (36). Although anti-Fc{epsilon}RII/CD23 Ab failed to induce ECP, we have previously reported that activation of neutrophils through this receptor down-modulated L-selectin from their surface (41). The involvement of galectin-3 in the allergen-induced release of ECP was demonstrated by the fact that preincubation of neutrophils with lactose, but not with glucose, abolished the ECP release by allergens (Fig. 4C).

Our results are exclusively due to neutrophils, and cannot be ascribed to a possible contamination by eosinophils for several reasons: 1) the two cell types had a different course of ECP release. As previously described (44, 54), eosinophils released ECP after 30 min of cell stimulation; neutrophils released protein only after 3–18 h. 2) In agreement with previous reports (54, 55), PAF failed to induce ECP release by eosinophils, whereas it induced ECP release by neutrophils. 3) LPS induced ECP release from eosinophils (44), which is in agreement with our results, but not from neutrophils. 4) Ag-, anti-IgE-, anti-IgE plus PAF-, anti-Fc{epsilon}RI-, and anti-galectin-3-dependent ECP production was observed in neutrophils, but not in eosinophils. In this regard, previous report have shown an absence of IgE-dependent ECP release from human eosinophils (8, 9, 10). 5) ECP mRNA was detected in unstimulated eosinophils (54), but not in unstimulated neutrophils. 6) CD66b is a specific marker for neutrophils (56) and we found that these cells (CD66b+) are in fact highly expressing ECP. 7) Finally, CLC is a marker of eosinophils and basophils but not of neutrophils (48, 49, 50, 51, 52) and we did not find its transcript in our neutrophil preparations. These findings are greatly in support of no contamination of eosinophils and/or basophils in our neutrophil samples.

We have previously described an IgE-dependent mechanism releasing elastase (39) and lactoferrin (unpublished data from our laboratory) from human neutrophils. These neutrophil granule contents have been described as stimuli for eosinophil activation, including ECP release (57, 58). For this reason, it can be speculated that the neutrophil may be an eosinophil-regulatory cell in IgE-mediated hypersensitivity inflammation. Based on our experiments, we hypothesize that ECP released by neutrophils has a biological role in IgE-induced reactions. We are carrying out additional studies to verify this hypothesis.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


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

1 This work was supported by grants from the Junta de Andalucia (Ayudas Grupos de Investigación), Fundación de la Sociedad Española de Alergia e Inmunología Clínica, Fundación Sanitaria Virgen Macarena, and Fundación Alergol, Spain. A.V. and P.C. were supported by fellowships from the Ministerio de Ciencia y Tecnología, and Fundación Alergol, Spain. R.E. is a recipient of a postdoctoral grant (Juan de la Cierva) (SAF2003-00200) from the Ministerio de Educación y Ciencia, Spain. J.M. is under the Programa de Intensificación de la Actividad Investigadora del Sistema Nacional de Salud. Back

2 Address correspondence and reprint requests to Dr. Javier Monteseirín, Servicio Regional de Inmunología y Alergia, Hospital Universitario Virgen Macarena, Sevilla, Spain, c/Asunción 27, 3° Izda. E-41011, Sevilla, Spain. E-mail address: fmonteserinm{at}meditex.es Back

3 J.M., A.V., and P.C. contributed equally to this work. Back

4 Abbreviations used in this paper: ECP, eosinophil cationic protein; PVDF, polyvinylidene difluoride; CLC, Charcot-Leyden crystal protein; PAF, platelet-activating factor. Back

Received for publication October 27, 2006. Accepted for publication June 4, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Venge, P., J. Byström. 1998. Eosinophil cationic protein. Int. J. Biochem. Cell Biol. 30: 433-437. [Medline]
  2. Venge, P., J. Byström, M. Carlson, L. Håkansson, M. Karawacizyk, C. Peterson, L. Sevéus, A. Trulson. 1999. Eosinophil cationic protein (ECP): molecular and biological properties and the use of ECP as a marker of eosinophil activation in disease. Clin. Exp. Allergy 29: 1172-1186. [Medline]
  3. Rosenberg, H. F., J. B. Domachowske. 2001. Eosinophils, eosinophil ribonucleases, and their role in host defense against respiratory virus pathogens. J. Leukocyte Biol. 70: 691-698. [Abstract/Free Full Text]
  4. Capron, M., H. L. Spiegelberg, L. Prin, H. Bennich, A. E. Butterworth, R. J. Pierce, M. A. Ouaissi, A. Capron. 1984. Role of IgE receptors in effector function of human eosinophils. J. Immunol. 132: 462-468. [Abstract]
  5. Capron, M., T. Jouault, L. Prin, M. Joseph, J. C. Ameisen, A. E. Butterworth, J. P. Papin, J. P. Kusnierz, A. Capron. 1986. Functional study of a monoclonal antibody to IgE Fc receptor (Fc{epsilon}RII) of eosinophils, platelets, and macrophages. J. Exp. Med. 164: 72-89. [Abstract/Free Full Text]
  6. Truong, M. J., V. Gruart, F. T. Liu, L. Prin, A. Capron, M. Capron. 1993. IgE-binding molecules (Mac-2/{epsilon}BP) expressed by human eosinophils. Implication in IgE-dependent eosinophil cytotoxicity. Eur. J. Immunol. 23: 3230-3235. [Medline]
  7. Gounni, A. S., B. Lamkhioued, K. Ochiai, Y. Tanaka, E. Delaporte, A. Capron, J. P. Kinet, M. Capron. 1994. High-affinity IgE receptor on eosinophils is involved in defence against parasites. Nature 367: 183-186. [Medline]
  8. Kaneko, M., M. C. Swanson, G. J. Gleich, H. Kita. 1995. Antigen-specific IgG1 and IgG3 through Fc{gamma}RII induce eosinophil degranulation. J. Clin. Invest. 95: 2813-2821. [Medline]
  9. Kita, H., M. Kaneko, K. R. Bartemes, D. A. Weiler, A. W. Schimming, C. Reed, G. J. Gleich. 1999. Does IgE bind to and activate eosinophils from patients with allergy?. J. Immunol. 162: 6901-6911. [Abstract/Free Full Text]
  10. Tomassini, M., A. Tsicopoulus, P. C. Tai, V. Gruart, A. B. Tonnel, L. Prin, A. Capron, M. Capron. 1991. Release of granule proteins by eosinophils from allergic and nonallergic patients with eosinophilia on immunoglobulin-dependent activation. J. Allergy Clin. Immunol. 88: 365-375. [Medline]
  11. Abu-Ghazaleh, R. I., S. L. Dunnette, D. A. Loegering, J. L. Checkel, H. Kita, L. L. Thomas, G. J. Gleich. 1992. Eosinophil granule proteins in peripheral blood granulocytes. J. Leukocyte Biol. 52: 611-618. [Abstract]
  12. Sur, S., D. G. Glitz, H. Kita, S. M. Kujawa, E. A. Peterson, D. A. Weiler, G. M. Kephart, J. M. Wagner, T. J. George, G. J. Gleich, K. M. Leiferman. 1998. Localization of eosinophil-derived neurotoxin and eosinophil cationic protein in neutrophilic leukocytes. J. Leukocyte Biol. 63: 715-722. [Abstract]
  13. Byström, J., R. C. Garcia, L. Håkansson, M. Karawajczyk, L. Moberg, J. Soukka, P. Venge. 2002. Eosinophil cationic protein is stored in, but not produced by, peripheral blood neutrophils. Clin. Exp. Allergy 32: 1082-1091. [Medline]
  14. Wilde, C. G., J. L. Snable, J. E. Griffith, R. W. Scott. 1990. Characterization of two azurophil granule proteases with active-site homology to neutrophil elastase. J. Biol. Chem. 265: 2038-2041. [Abstract/Free Full Text]
  15. Metso, T., P. Venge, T. Haahtela, C. G. B. Peterson, L. Sevéus. 2002. Cell specific markers for eosinophils and neutrophils in sputum and bronchoalveolar lavage fluid of patients with respiratory conditions and healthy subjects. Thorax 57: 449-451. [Abstract/Free Full Text]
  16. Amin, K., J. Rinne, T. Haahtela, M. Simola, C. G. B. Peterson, G. M. Rooman, H. Malmberg, P. Venge, L. Sevéus. 2001. Inflammatory cell and epithelial characteristics of perennial allergic and nonallergic rhinitis with a symptom history of 1 to 3 years’ duration. J. Allergy Clin. Immunol. 107: 249-257. [Medline]
  17. Heldal, K. K., A. S. Halstensen, J. Thorn, P. Djupesland, I. Wouters, W. Eduard. 2003. Upper airway inflammation in waste handlers exposed to bioaerosols. Occup. Environ. Med. 60: 444-450. [Abstract/Free Full Text]
  18. Ying, S., Q. Meng, S. J. Smith, M. Larche, D. S. Robinson, A. B. Kay. 2002. Methods for identifying human eosinophils in blood and tissue. ACI Int. 14: 64-71.
  19. Azevedo, I., J. de Blic, B. B. Vargaftig, M. Bachelet, P. Scheinmann. 2001. Increased eosinophil cationic protein levels in bronchoalveolar lavage from wheezy infants. Pediatr. Allergy Immunol. 12: 65-72. [Medline]
  20. Marguet, C., T. P. Dean, J. P. Basuyau, J. O. Warner. 2001. Eosinophil cationic protein and interleukin-8 levels in bronchial lavage fluid from children with asthma and infantile wheeze. Pediatr. Allergy Immunol. 12: 27-33. [Medline]
  21. Benson, M., I. L. Strannegård, O. Strannegård, G. Wennergren. 2000. Topical steroid treatment of allergic rhinitis decreases nasal fluid Th2 cytokines, eosinophils, eosinophil cationic protein, and IgE but has no significant effect on IFN-{gamma}, IL-1beta, TNF-{alpha}, or neutrophils. J. Allergy Clin. Immunol. 106: 307-312. [Medline]
  22. Barbato, A., C. Panizzolo, M. Gheno, L. Sainati, E. Favero, D. Faggian, F. Giusti, L. Pesscolderungg, M. La Rosa. 2001. Bronchoalveolar lavage in asthmatic children: evidence of neutrophil activation in mild-to-moderate persistent asthma. Pediatr. Allergy Immunol. 12: 73-77. [Medline]
  23. Jatakanon, A., C. Vasuf, W. Maziak, S. Lim, K. F. Chung, P. J. Barnes. 1998. Neutrophilic inflammation in severe persistent asthma. Am. J. Respir. Crit. Care Med. 157: 403-409. [Medline]
  24. Leigh, R., J. Belda, M. M. Kelly, G. Cox, D. L. Squillace, G. J. Gleich, F. E. Hargreave. 2000. Eosinophil cationic protein relates to sputum neutrophil counts in healthy subjects. J. Allergy Clin. Immunol. 106: 593-594. [Medline]
  25. Henson, P. M., L. C. Borish. 1995. Neutrophil mediators in asthma. W. W. Busse, and S. T. Holgate, eds. Asthma and Rhinitis 367-382. Blackwell Scientific Publications, Boston.
  26. Carlson, M., L. Håkansson, C. Peterson, G. Stålenheim, P. Venge. 1991. Secretion of granule proteins from eosinophils and neutrophils is increased in asthma. J. Allergy Clin. Immunol. 87: 27-33. [Medline]
  27. Lee, T. H., T. Nagakura, M. J. Wallport, A. B. Kay. 1982. Identification and partial characterization of an exercise-induced neutrophil chemotactic factor in bronchial asthma. J. Clin. Invest. 69: 889-899. [Medline]
  28. Nagy, L., T. H. Lee, A. B. Kay. 1982. Neutrophil chemotactic activity in antigen-induced late asthmatic reactions. N. Engl. J. Med. 306: 497-501. [Abstract]
  29. Boshetto, P., C. E. Mapp, G. Picotti, L. M. Fabbri. 1989. Neutrophils and asthma. Eur. Respir. J. 6: 456s-459s.
  30. Fabbri, L. M., P. Boshetto, E. Zocca, G. Milani, F. Pivirotto, M. Plebani, A. Burlina, B. Licata, C. E. Mapp. Bronchoalveolar neutrophilia during late asthmatic reactions induced by toluene diisocyanate. Am. Rev. Respir. Dis. 136: 36-42.
  31. Metzger, W. J., H. B. Richerson, K. Worden, M. Monick, G. W. Hunninghake. 1986. Bronchoalveolar lavage of allergic asthmatic patients following allergen bronchoprovocation. Chest 89: 477-483. [Medline]
  32. Gounni, A. S., B. Lamkhioued, L. Koussih, Ch. Ra, P. M. Renzi, Q. Hamid. 2001. Human neutrophils express the high affinity receptor for IgE (Fc{epsilon}RI): role in asthma. FASEB J. 5: 940-949.
  33. Yamaoka, K. A., M. Arock, F. Issaly, N. Dugas, L. Le Goff, J. P. Kolb. 1996. Granulocyte macrophage colony stimulating factor induces Fc{epsilon}RII/CD23 expression on normal human polymorphonuclear neutrophils. Int. Immunol. 8: 479-490. [Abstract/Free Full Text]
  34. Vella, A., P. Bellavite, A. Adami, R. Ortolani, G. Benoni, A. Carletto, D. Biasi, P. Caramaschi, G. Tridente. 1999. Expression of Fc{epsilon}RII/CD23 on human neutrophils isolated from rheumatoid arthritis patients. Inflammation 23: 471-479. [Medline]
  35. Truong, M. J., V. Gruart, J. P. Kusnierz, J. P. Papin, S. Loiseau, A. Capron, M. Capron. 1993. Human neutrophils express immunoglobulin E (IgE)-binding proteins (Mac2/{epsilon}BP) of the S-type lectin family: role in IgE-dependent activation. J. Exp. Med. 117: 243-248.
  36. Yamaoka, A., I. Kuwabara, L. G. Frigeri, F. T. Liu. 1995. A human lectin, galectin-3 ({epsilon}BP/Mac2), stimulates superoxide production by neutrophils. J. Immunol. 154: 3479-3487. [Abstract]
  37. Monteseirín, J., M. J. Camacho, R. Montaño, E. Llamas, M. Conde, M. Carballo, P. Guardia, J. Conde, F. Sobrino. 1996. Enhancement of antigen-specific functional responses by neutrophils from allergic patients. J. Exp. Med. 183: 2571-2579. [Abstract/Free Full Text]
  38. Monteseirín, J., I. Bonilla, M. J. Camacho, J. Conde, F. Sobrino. 2001. IgE-dependent release of myeloperoxidase by neutrophils from allergic patients. Clin. Exp. Allergy 31: 889-892. [Medline]
  39. Monteseirín, J., I. Bonilla, M. J. Camacho, P. Chacon, A. Vega, A. Chaparro, J. Conde, F. Sobrino. 2003. Specific antigens enhance elastase release in stimulated neutrophils from asthmatic patients. Int. Arch. Allergy Immunol. 131: 174-181. [Medline]
  40. Monteseirín, J., P. Chacon, A. Vega, R. El Bekay, M. Alvarez, G. Alba, M. Conde, J. Jimenez, J. A. Asturias, A. Martínez, et al 2004. Human neutrophils synthesize IL-8 in an IgE-mediated activation. J. Leukocyte Biol. 76: 692-699. [Abstract/Free Full Text]
  41. Monteseirín, J., P. Chacón, A. Vega, H. Sánchez-Hernández, J. A. Asturias, A. Martínez, P. Guardia, R. Pérez-Cano, J. Conde. 2005. L-selectin expression on neutrophils from allergic patients. Clin. Exp. Allergy 35: 1204-1213. [Medline]
  42. National Heart, Lung, and Blood Institute 1998. Global strategy for asthma management and prevention NIH Publication No. 96-3659B, Bethesda.
  43. Hansel, T. T., I. J. De Vries, T. Iff, S. Rihs, M. Wandzilak, S. Betz, K. Blaser, C. Walker. 1991. An improved immunomagnetic procedure for the isolation of highly purified human blood eosinophils. J. Immunol. Methods 145: 105-110. [Medline]
  44. Plotz, S. G., A. Lentschat, H. Behrendt, W. Plotz, L. Hamann, J. Ring, E. T. Rietschel, H. D. Flad, A. J. Ulmer. 2001. The interaction of human peripheral blood eosinophils with bacterial lipopolysaccharide is CD14 dependent. Blood 97: 235-241. [Abstract/Free Full Text]
  45. Mansfield, M. A.. 1995. Rapid immunodetection on polyvinylidene fluoride membrane blots without blocking. Anal. Biochem. 229: 140-143. [Medline]
  46. Carballo, M., G. Marquez, M. Conde, J. Martin-Nieto, J. Monteseirin, J. Conde, E. Pintado, F. Sobrino. 1999. Characterization of calcineurin in human neutrophils: inhibitory effect of hydrogen peroxide on its enzyme activity and on NF-{kappa}B DNA binding. J. Biol. Chem. 274: 93-100. [Abstract/Free Full Text]
  47. Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159. [Medline]
  48. Ackerman, S. J., S. E. Correte, H. F. Rosenberg, J. C. Bennet, D. M. Mastriani, A. Nicholson-Weller, P. F. Weller, D. T. Chin, D. G. Tenen. 1993. Molecular cloning and characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase): similarities to IgE binding proteins and S-type animal lectin superfamily. J. Immunol. 150: 456-468. [Abstract]
  49. Gomolin, H. I., Y. Yamaguchi, A. V. Paulpillai, L. A. Dvorak, S. J. Ackerman, D. G. Tenen. 1993. Human eosinophil Charcot-Leyden crystal protein: cloning and characterization of a lysophospholipase gene promoter. Blood 82: 1868-1874. [Abstract/Free Full Text]
  50. Dvorak, A. M., L. Letourneau, G. R. Login, P. F. Weller, S. J. Ackerman. 1989. Ultrastructural localization of the Charcot-Leyden crystal protein (lysophospholipase) to a distinct crystalloid-free granule population in mature human eosinophils. Blood 72: 150-158.
  51. Leiferman, K. M., G. J. Gleich, G. M. Kephart, H. S. Haugen, K. Hisamatsu, D. Proud, L. M. Lichtenstein, S. J. Ackerman. 1986. Differences between basophils and mast cells: failure to detect Charcot-Leyden crystal protein (lysophospholipase) and eosinophil major basic protein in human mast cells. J. Immunol. 136: 852-855. [Abstract]
  52. Golightly, L. M., L. L. Thomas, A. M. Dvorak, S. J. Ackerman. 1992. Charcot-Leyden crystal protein in the degranulation and recovery of activated basophils. J. Leukocyte Biol. 51: 386-392. [Abstract]
  53. Frigeri, L. G., F. T. Liu. 1992. Surface expression of functional IgE binding protein, an endogenous lectin, on mast cells and macrophages. J. Immunol. 148: 861-867. [Abstract]
  54. Simon, H. U., M. Weber, E. Becker, Y. Zilberman, K. Blaser, F. Levi-Schaffer. 2000. Eosinophils maintain their capacity to signal and release eosinophil cationic protein upon repetitive stimulation with the same agonist. J. Immunol. 165: 4069-4075. [Abstract/Free Full Text]
  55. Turner, N. C., L. J. Wood, M. Foster, T. Gueremy. 1994. Effects of PAF, FMLP and opsonized zymosan on the release of ECP, elastase and superoxide from human granulocytes. Eur. Respir. J. 7: 934-940. [Abstract]
  56. Borregaard, N., J. B. Couland. 1997. Granules of the human neutrophilic polymorphonuclear leukocyte. Blood 89: 3503-3521. [Free Full Text]
  57. Liu, H., S. C. Lazarus, G. H. Caughey, J. V. Fahy. 1999. Neutrophil elastase and elastase-rich cystic fibrosis sputum degranulate human eosinophils in vitro. Am. J. Physiol. 276: L28-L34. [Medline]
  58. Thomas, L. L., W. Xu, T. T. Ardon. 2002. Immobilized lactoferrin is a stimulus for eosinophil activation. J. Immunol. 169: 993-999. [Abstract/Free Full Text]



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