Published online before print June 3, 2004
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,1
* Departamento de Medicina, Servicio de Inmunología y Alergia, Hospital Universitario Virgen Macarena and
Clínica Sagrado Corazón, Sevilla, Spain;
Bial-Aristegui, Departamento R&D, Bilbao, Spain; and
Departamento de Bioquímica Médica y Biología Molecular, Universidad de Sevilla, Spain
1 Correspondence: Dpto. Bioquímica Médica y Biología Molecular, Facultad de Medicina, Universidad de Sevilla, 41009-Sevilla, Spain. E-mail: fsobrino{at}us.es
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Key Words: anti-IgE • ROS • Ca2+ • calcineurin
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-)], microbial toxins, and other conditions that disturb homeostasis [1
, 4
, 11
12
13
14
]. As accumulation of neutrophils in tissues is a characteristic feature of inflammation, this capacity to produce IL-8 suggests that these cells can, by themselves, enhance recruitment of new neutrophils at local inflammatory sites. Thus, close regulation of neutrophil migration is necessary to control inflammatory reactions. Excessive production of IL-8 may be involved in the pathogenesis of various types of inflammatory reaction. Moreover, various cytokines and agents have been reported to inhibit IL-8 production. Cytokines such as IL-4 [15
], IL-10 [16
], transforming growth factor-ß, and interferon-ß [17
] counteract the induction of IL-8 production by IL-1, TNF-, or LPS. Immunomodulatory drugs such as glucocorticoids [18
] and vitamin D3 [19
] also act as suppressors of IL-8 production. An immunosuppressive drug, cyclosporin A (CsA), has been reported to suppress the induction of IL-8 mRNA in human T lymphocytes [20
].
Although eosinophils and lymphocytes have become more strongly associated with allergic diseases, peripheral blood neutrophils also show markers of activation during active asthma [21
] and after exercise-induced asthma [22
]. Previous studies have demonstrated the presence on human neutrophils of the three forms of the immunoglobulin (Ig)E receptor, Fc
RI [23
24
25
], FcRII/CD23 [26
27
28
], and galectin-3 [29
30
31
]. We have previously shown that specific antigens were able to activate functional responses of neutrophils from allergic patients sensitized to antigens of the same type as those that produce clinical allergic symptoms [32
33
34
35
]. Our group described the specific binding antigens on the surface of neutrophils, using flow cytometry and microscopy analysis [33
]. Other groups [24
, 27
, 29
, 31
] have demonstrated the presence of IgE molecules bound on the surface of neutrophils. Furthermore, we have shown the specific IgE bound on the surface of neutrophils from allergic patients [32
]. In the present work, we set out to analyze whether the attachment of cellular IgE molecules with anti-IgE/specific antigens was able to regulate IL-8 production and its gene expression in human neutrophils from patients sensitized to the antigens. Additionally, we have investigated whether reactive oxygen species (ROS) and calcium/calcineurin could participate in that regulation. In this context, the evidence here shown points to neutrophils as cells implicated in the IgE-mediated inflammatory and pathological states, such as in allergic diseases.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of polymorphonuclear leukocytes and cell culture
The group studied included adult atopic patients with bronchial asthma and healthy adult nonatopic volunteer controls. The asthmatic patients had positive skin-prick tests (Bial-Aristegui) and specific IgE (HYTEC 288, IZASA, Barcelona, Spain) to at least one common antigen (house dust mites and pollens). They received neither treatment nor specific hyposensitization and were not allowed to take any bronchodilators within the 8 h before challenge of neutrophils in vitro. Oral bronchodilators were withheld for 24 h, and none of the subjects had taken corticosteroids, cromolyn sodium, or nedocromil sodium in the previous week. The healthy controls had no history of allergy or bronchial symptoms and had negative skin-prick tests and specific IgE to a battery of inhalant antigens. The Hospital Universitario Virgen Macarena (Sevilla, Spain) Ethics Committee approved the study, and each subject gave informed consent.
Human neutrophils were purified from fresh-drawn, heparinized (10 U/ml) venous blood as follows: The blood (20 ml) was mixed with 1.5 ml 10% Dextran T 500 (final concentration of 0.7%), dissolved in phosphate-buffered saline (PBS). The supernatant fraction was centrifuged through a Ficoll-Hypaque. The neutrophil-rich pellet was then removed with a plastic pipette, and red cells were eliminated by one hypotonic lysis in water [32
33
34
35
, 38
]. Neutrophils were washed twice with PBS, resuspended in RPMI-1640 medium supplemented with 10% fetal serum albumin, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B, and cultured at 37°C under a humidified air atmosphere containing 5% CO2 and 95% O2. For further purification, contaminating eosinophils were depleted with mouse anti-human CD9 Ab (Immunotech-IZASA) and goat anti-mouse IgG micromagnetic beads (Miltenyi Biotech, Bergisch-Gladbach, Germany). The purity of neutrophils was, on average, >99%. Eosinophil contamination was 
1%.
For stimulation treatments, the cells were incubated with allergens, IgG, or anti-IgE for the indicated times at 37°C. The inhibitors EGTA, TFP, and PDTC were added 60 min and CsA, DPI, TEMPO, and HMAP, 30 min prior to stimulation. None of these reagents affected the viability of the cells at the concentrations used. Trypan blue exclusion showed greater than 96% viability in neutrophils. In all cases, the cells were maintained in culture for the same time in the presence or absence of stimuli.
Dissociation of neutrophil-bound Igs and immunoblotting methods
Ig molecules were dissociated from the surface of neutrophils as described previously [32
]. Briefly, after isolation, cells (1x108 cells) 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 (GVB; 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 g for 10 min. Supernatants were collected and neutralized with 1 N NaOH and used for specific IgE and IgG analyses. Antigenic extracts were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions and transferred to polyvinylidene difluoride (PVDF) membranes using a semi-dry device (Bio-Rad, Richmond, VA). After transference and blocking, membranes were probed for 12 h at room temperature with previously obtained IgE-containing supernatants, which were diluted 1:7 in GVB. After washing, filters were incubated with HRP-conjugated anti-human IgE, diluted 1:750 in GVB buffer for 1 h at room temperature, and then washed. Immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) assays as described previously [38
].
Analysis of IL-8 release
Cells were dispensed in 24-well tissue-culture plates (Nunc, Roskilde, Denmark) at a density of 2 x 106 cells/ml and cultured in the absence or presence of additions at the times and doses indicated in each case. Supernatants were harvested and stored at –80°C until measuring and thawed only once for cytokine detection. IL-8 in the supernatants was measured using the enzyme-linked immunosorbent assay (ELISA) kit obtained from Bender MedSystems (Vienna, Austria)
RNA extraction, reverse transcriptase (RT), and conventional PCR
As RT-PCR is a sensitive method, it was important to isolate RNA for extremely pure neutrophils to avoid false-positive results as a result of contamination with eosinophils. To this end, neutrophils for RT-PCR were purified by the micromagnetic beads system as described above, but the procedure was repeated three more times using 4 µg/ml CD9 Ab each time. Effective removal of eosinophils was evaluated by flow cytometry [fluorescein-activated cell sorter analysis (EPICS Elite, Coulter-IZASA)] with anti-CD9-RD1 Ab (Coulter-IZASA). The contaminating eosinophils were between 0.001% and 0.004% [39
, 40
]. Total RNA was isolated from 5 x 106 neutrophils using the RNA isolation kit RNeasy (Quiagen, IZASA), following the manufacturer’s protocols. Total RNA (1 µg) was reverse-transcribed to first-strand cDNA using random primers. The first-strand cDNA was amplified with primer sets for human IL-8 by conventional PCR [41
]. Aliquots taken after 30 cycles (20 µl) were electrophoresed on 1.5% agarose gels. The IL-8 0.2-kb and ß-actin 0.5-kb PCR products were visualized on ethidium bromide-stained agarose gels and photographed under UV light.
Real-time PCR
After total RNA extraction, 750 ng was subsequently used for cDNA synthesis as described above. Real-time PCR was performed in an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) using SYBR Green PCR Master Mix (Qiagen, Izasa, Portugal) and the thermocycler conditions recommended by the manufacturer. PCR reactions were performed in triplicate in a total volume of 25 µl containing 2 µl of the RT reaction. Each sample was analyzed for rRNA 28S to normalize for RNA input amounts and to perform relative quantifications. Primers were designed using the computer program Primer Express (Applied Biosystems). Primers (IL-8 forward: 5'-ATTAGCCACCATCTTACCTCACAGT-3'; IL-8 reverse: 5'-GTGCTTCCACATGTCCTCACA-3'; rRNA 28S foward: 5'-GTGACGCGCATGAATGGA-3'; rRNA 28S reverse: 5'-CCCTTGGCTGTGGTTTCG-3') were generated and used to amplify 50-bp and 100-bp fragments, respectively. Melting curve analysis showed a single sharp peak with the expected temperature melting (Tm) for all samples. For the relative quantification of gene expression, the comparative threshold cycle (CT) method was used as described in User Bulletin 2 for ABI Prism 7700 sequence detection system. CT represents the PCR cycle at which an increase in reporter fluorescence above a background signal can first be detected (10 times the standard deviation of the baseline). First, internal control (rRNA28S) CT values were subtracted from the gene-of-interest CT values to derive a 
CT value. The relative expression of the gene-of-interest was then evaluated using the expression 2–CT, where the value for CT was obtained by subtracting the CT of the calibrator from each CT, using the mean of the control (t=0) as the calibrator.
Statistical analysis
Data are expressed as means ± SEM. A Student’s unpaired t-test or one-way ANOVA was used to make comparisons between groups. A P value <0.05 was considered significant.
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Figure 1. Dose-dependent effect of anti-IgE Ab and specific antigen on IL-8 production from human neutrophils. (A) Neutrophils (106 cells/0.5 ml) obtained from allergic patients were incubated with increased amounts of anti-IgE (-IgE) for 18 h (•). The control goat IgG is shown ( ). Human IL-8 content in the culture supernatants was determined using an ELISA assay. (B) Neutrophils (106 cells/0.5 ml) from allergic patients who were specifically sensitized to G3 were incubated with G3 (•) or D1( ), to which the patients were not sensitized. The values shown are the mean ± SEM from four independent assays in which each measurement was performed in duplicate for each group.
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Figure 2. Time-course of IL-8 release by neutrophils stimulated with anti-IgE Ab, fMLP, and LPS. Neutrophils isolated from allergic patients were incubated at 37°C with 15 µg/ml anti-IgE (-IgE), 100 nM fMLP, and 0.5 µg/ml LPS. At indicated times, incubation medium was collected for IL-8 measurement. Plotted values are the mean ± SEM from five assays in which each measurement was performed in duplicate. Statistical significance (vs. control at each time): *, P < 0.001; **, P = 0.006; ***, P = 0.007;  , P = 0.004.
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1.5-fold accumulation of the IL-8 mRNA after 3 h of exposure, with a maximun effect at 18 h (fivefold induction).
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Figure 3. IL-8 mRNA expression in anti-IgE Ab-stimulated human neutrophils from allergic patients. Neutrophils (5x106 cells/1 ml) were incubated in the presence or absence of 15 µg/ml anti-IgE (-IgE) for the indicated times. Total RNA was isolated and reverse-transcribed, and the resulting cDNA was amplified by conventional PCR (A) or by real-time PCR (B). In conventional PCR, ß-actin was used to normalize the amount of RNA. For real-time PCR, we show the average fold induction ± SEM from two separate measurements.
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We have found (Fig. 4
) immunoreactive bands only with T9 antigen extracts (Lane 1), and no reactivity was detected when the eluted supernatants were incubated with other antigens extracts (i.e., D1), to which the patients were not sensitized (Lane 3), or when the supernatants were from cells without treatment (Lane 2). Similar results were previously obtained for other antigens tested [32
]. In the present case, the apparent molecular weight of the two major observed bands from T9 extracts, which bind to IgE eluted from the neutrophil surface, were of 36 and 40 kDa. Other minor bands with molecular weight in a range of 49–60 kDa were also detected. The diversity of the size of these bands could represent different T9 antigenic components recognized by neutrophils, with potential capacity to activate these cells (e.g., releasing IL-8 cytokine). These results suggest the existence of specific IgE bound to the cellular membrane of human neutrophils from allergic patients.
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Figure 4. Reactivity of specific IgE (eluted from the neutrophil cell surface) to allergenic extracts. The extracts of specific antigens were resolved by SDS-PAGE and transferred to PVDF membranes. Then, membranes were probed with different supernatants: Lane 1, T9 extract was probed with supernatants from neutrophils of a patient with T9-specific IgE after the treatment to dissociate IgE from the cell surface; lane 2, T9 extract was probed with supernatants from neutrophils of a patient with T9-specific IgE without the treatment to dissociate IgE from the cell surface; lane 3, D1 extract was probed with supernatants from neutrophils of a patient with T9-specific IgE after the treatment to dissociate IgE from the cell surface. After washing, membranes were incubated with HRP-conjugated anti-human IgE and revealed with an ECL assay. Molecular weights (M.W.) are shown.
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Table 1. Ca2+ Dependence of IL-8 Release by -IgE-Stimulated Human Neutrophils
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Figure 5. ROS dependence of anti-IgE Ab-induced IL-8 production by human neutrophils. Cells from allergic patients were preincubated with the following agents: HMAP (500 µM), DPI (10 µM), or TEMPO (100 µM) for 30 min or PDTC (100 µM) for 60 min, as indicated. Then, anti-IgE (-IgE; 15 µg/ml) was added over 20 h (A) or 3 h (B). IL-8 release (A) was assayed as indicated in Materials and Methods. IL-8 mRNA (B) was analyzed by conventional PCR. (A) Values shown are the mean ± SEM from three separate experiments, in which each measurement was performed in duplicate. (B) Data shown are representative of another three separate experiments.
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Figure 6. CsA suppressed the IL-8 release from activated human neutrophils. (A) Neutrophils from allergic patients were preincubated for 30 min with the indicated concentration of CsA before the addition of 15 µg/ml anti-IgE (-IgE) and were then incubated for another 20 h. The amount of secreted IL-8 in the cell-free supernatants was analyzed by ELISA. (B) Neutrophils from G3-sensitized patients were incubated in the presence or absence of 1 µg/ml CsA for 30 min before the addition of stimulus: 15 µg/ml anti-IgE, 0.5 µg/ml LPS, or 50 µg/ml G3 for another 20 h. The values shown are the mean ± SEM from three separate experiments.
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Figure 7. Effect of increasing amounts of CsA on the expression of IL-8 mRNA. Neutrophils from allergic donors were incubated in the presence or absence of CsA for 30 min or 2.5 mM EGTA for 1 h, and further, 15 µg/ml anti-IgE (-IgE) was added. Total RNA was extracted after 3 h of incubation, reverse-transcribed, and amplified by conventional PCR. mRNAs of IL-8 and ß-actin are shown. Data presented are representative of three experiments performed in similar conditions.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Real-time PCR analysis of RNA isolated from highly purified peripheral blood neutrophils demonstrated that the levels of IL-8 mRNA synthesized by allergic patients were low in untreated neutrophils after 18 h of incubation, whereas treatment of cells with anti-IgE Ab induced a significant time-dependent increase in the level of specific mRNA, which was significant at 3 h of treatment and reached maximum levels at 18 h. In agreement with present data, purified eosinophil granule major basic protein increased IL-8 mRNA levels in neutrophils after 3 h of incubation [56 ]. Other agents, such as thapsigargin (an inhibitor of microsomal Ca2+ pumps), caused a rapid induction (within 5 min) of IL-8 mRNA [46 ].
IL-8 is a classical, adenosine-uridine element-rich mRNA [57 ]. These elements are believed to be associated with message instability, translational efficiency, and rapid turnover. In this regard, the increase in IL-8 mRNA level observed after IgE-dependent stimulation of human neutrophils could result from transcriptional and/or stabilization activities of the mRNA. Thus, IgE-dependent treatment of neutrophils stimulated IL-8 expression at mRNA and protein levels.
Previously, we have shown that in neutrophils from allergic patients, Ca2+ levels increased when the cells were challenged with the specific antigens or anti-IgE Ab [33 ]. Other studies demonstrated that although unstimulated peripheral blood neutrophils produced minimal levels of IL-8, the Ca2+ ionophore A23187 increased IL-8 to levels comparable with those found in exudative neutrophils isolated from an inflammatory site in vivo [45 ]. The data suggested that elevation of intracellular [Ca2+] may be an important physiologic signal regulating production of the inflammatory cytokine IL-8 by neutrophils. The studies presented in the current report extend those observations by demonstrating that incubation with antigen or anti-IgE Ab of freshly isolated peripheral blood neutrophils from allergic patients caused a dose-dependent increase in the production of the chemokine IL-8 to levels comparable with those previously observed in exudative neutrophils [45 ]. The secretion of synthesized IL-8 was inhibited by addition of EGTA, suggesting that the influx of Ca2+ was necessary not only for the synthesis of IL-8 but its secretion as well. These findings are similar to the case of thapsigargin-induced IL-8 production in human neutrophils [46 ]. TFP, a calmodulin (CaM) inhibitor, inhibits IgE-dependent liberation of IL-8 by neutrophils from allergic patients. CaM is involved in many fundamental signaling pathways, activating the function of various kinases (CaM kinases I and II, myosin light-chain kinase), phosphatases (calcineurin), ion channels (plasma membrane Ca2+ pump), and other cytosolic enzymes, such as phosphodiesterase, adenylate cyclase, and nitric oxide synthetase [58 ]. Thus, it is not surprising that inhibitors of CaM typically inhibit cell activation processes. CaM inhibitors have been shown to inhibit neutrophil oxidative burst, membrane-activated complex-1 up-regulation, secondary granule release, cell migration, cell motility [59 , 60 ], and in our case, IgE-dependent IL-8 liberation. In this study, we demonstrate that TFP or EGTA decreased the IL-8 production in human neutrophils. Both data are in agreement with the role of calcium-dependent pathways in the IgE-dependent IL-8 liberation by neutrophils.
Another goal of this study concerned the presumptive role of the ROS in the IgE-dependent IL-8 production by human neutrophils. We previously reported an enhanced oxidative burst after stimulation with specific antigens or anti-IgE Ab in neutrophils from allergic patients [33
, 34
]. Furthermore, several lines of evidence have indicated the participation of oxidants in the IL-8 gene expression in different cell types, such as homocysteine-mediated IL-8 expression in human monocytes [47
] or the H2O2/TNF--induced IL-8 synthesis by human umbilical vein epithelial cells [48
]. In agreement with these reports, our results indicate that the effects of anti-IgE Ab were primarily mediated by ROS through NADPH oxidase, as inhibitors of this enzyme, such as DPI and HMAP, and free radical scavengers, such as PDTC or TEMPO, abolished IL-8 synthesis and secretion.
Previously, we described a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+ and redox status [38 , 54 ]. For this reason, in the current study, we also explored the potential role of this intracellular protein in the stimulatory effect of an IgE-dependent treatment toward IL-8 production in neutrophils.
Here, we present evidence that CsA, an immunosuppressive drug that binds to specific immunophilin and interferes with the activation of the Ca2+/CaM-dependent serine/threonine phosphatase calcineurin (or protein phosphatase 2B) [61 ], inhibits, in a dose-dependent manner, the IgE-dependent induction of IL-8 mRNA and protein in neutrophils from allergic patients. Present data are in accord with earlier reports that IL-8 induction by the CD28 costimulatory pathway in Jurkat cells is sensitive to CsA [62 ] and that the thapsigargin-induced up-regulation of the IL-8 gene in neutrophils is inhibited by CsA [46 ].
Besides being a potent chemoattractant and stimulus for neutrophils [1 , 7 ], IL-8 is also a chemoattractant for T lymphocytes [5 ] and for IL-5-primed eosinophils [63 ], as well as a stimulus for basophils primed by IL-3 [64 ]. Thus, IgE-induced IL-8 production by neutrophils could contribute to inflammatory events characteristic of chronic asthma. However, the effect of IgE on neutrophil IL-8 release may be even more relevant to the pathophysiology of acute asthma. In addition to promoting an abnormally high neutrophil influx, IL-8 release could prolong neutrophil activation in this pathological condition in an autocrine-like manner, and the resulting increased release of elastase and other mediators [65 ] could exacerbate changes such as formation of the mucus plugs observed in fatal asthma. Furthermore, levels of IL-8 were elevated during the acute exacerbation of bronchial asthma [66 ] or challenge with bronchial antigens [67 ]. In summary, we show evidence that an IgE-dependent mechanism is able to release IL-8 and up-regulate IL-8 mRNA expression by neutrophils from allergic patients. Furthermore, these events are mediated through a Ca2+, ROS, and the calcineurin-dependent pathway.
Received September 24, 2003; accepted May 5, 2004.
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