Abstract
Reactive oxygen species (ROS) from eosinophils are known to cause tissue damage in allergic inflammation. CC chemokines, especially eotaxin and regulated on activation, normal T‐cell expressed and secreted (RANTES), are involved not only in chemotaxis but also in eosinophil activation, such as ROS production. It has been shown that eosinophils from allergic patients are not functionally equivalent to those from normal subjects. In the present study, the characteristics of chemokineprimed ROS production in eosinophils from allergic patients and normal controls were compared.
After pretreatment with chemokines, eosinophils were stimulated with calcium ionophore A23187. ROS production by eosinophils was measured using luminoldependent chemiluminescence.
Both RANTES and eotaxin exhibited a priming effect on calcium ionophoreinduced ROS production from eosinophils. Despite there being no difference in expression of CC chemokine receptor 3, the priming effect of RANTES and eotaxin was significantly enhanced in eosinophils from the patients. Interleukin‐5 further enhanced the priming effect of chemokines in eosinophils from normal subjects, but not those from allergic subjects.
The present results suggest an upregulated response to chemokines in eosinophils from allergic patients, and that interleukin‐5 can induce a similar phenotype to that found in vivo in allergic patients.
- CC chemokine receptor 3
- eosinophils
- eotaxin
- interleukin‐5
- reactive oxygen species
- regulated on activation
- normal T‐cell expressed and secreted
This study was supported by grantsinaid for Scientific Research from the Ministry of Education and Science, and the Ministry of Health and Welfare (both Tokyo, Japan).
One characteristic feature of allergic disease is tissue inflammation, involving the activation of T‐lymphocytesand eosinophils 1. The severity of allergic disease is influenced bythe degree of eosinophil activation. During the process ofallergic inflammation, eosinophils migrate into tissues andrelease toxic granule proteins and reactive oxygen species (ROS), leading to tissue damage 2.
ROS production is elicited by several stimuli, such as immunoglobulins (Igs) and cytokines 3. It has been previously reported that the signal from adhesion molecules plays a critical role in ROS production by eosinophils 4. TheCC chemokines, especially eotaxin and regulated on activation, normal T‐cell expressed and secreted (RANTES), possess a selective chemotactic activity for eosinophils. Besides chemotaxis, these chemokines are involved in eosinophil activation. Indeed, it has been shown recently that chemokines prime ROS production by eosinophils 5, 6.
It has been shown that eosinophils from allergic patients are not equivalent in effector function to those from normalsubjects 7–9. However, the different response of eosinophils to chemokines has not been fully elucidated. Therefore, in thepresent paper, comparative studies were performed inallergic patients and normal subjects regarding the primingeffects of chemokines on ROS production from eosinophils.
Materials and methods
Subjects
Venous blood was drawn from 12 healthy nonallergic adults (age 18–40 yrs, mean 27.3 yrs; four females) and from 15 patients with allergic diseases of the respiratory tract (23–32 yrs, mean 25.1 yrs; five females). Age and sex distribution were not significantly different between normal subject and patient groups. All subjects gave informed consent, and the study was carried out according to the principles of the Declaration of Helsinki. None of the subjects had received either any medication for ≥24 h or steroids for ≥2 weeks before blood collection. Normal subjects were defined on thebasis of a lack of a clinical history of allergy or other similar diseases. All patients had allergic asthma and/or allergic rhinitis, IgE concentrations of >400 International Units (IU)·mL−1 and an IgE radioallergosorbent test result of higher than class 3 against at least one of the common airborne allergens, such as house dust mite, pollens or fungi. The numbers of patients with asthma and allergic rhinitis were seven and 11, respectively (three had both asthma and allergic rhinitis). Asthmatic patients participating in the present study met the American Thoracic Society's definition of asthma. Allpatients with allergic rhinitis showed symptoms at the time of blood collection (nasal congestion, sneezing, rhinorrhoea, itchy eyes, etc.). The eosinophil counts in the peripheral blood of patients were significantly higher than those of normal subjects (683.3±374.0 versus 126.0±80.4 cells·mm−3; p<0.01).
Eosinophil isolation
Eosinophils were isolated from heparinised venous blood using a modified CD16 negative selection method, as previously described 10. In brief, cells obtained from the buffy coat were incubated with antiCD16, antiCD3, antiCD20 and antiCD14 monoclonal antibodies (mouse IgG; Nichirei, Tokyo, Japan), and subsequently reacted with antimouse IgG magnetic beads (Dynal, Oslo, Norway). CD16, CD3, CD20 and CD14negative eosinophils were obtained using a magnetic cellsorting system (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the eosinophils was >97%.
Luminoldependent chemiluminescence
ROS production from eosinophils was examined by means of luminoldependent chemiluminescence 5. Previously, an apparent effect of eotaxin and RANTES on eosinophil oxidative metabolism was found after 15min incubation 5, 6. Thus purified eosinophils (1×106 cells·mL−1) were suspended in Roswell Park Memorial Institute (RPMI) 1640 medium and incubated with 1–100 nM eotaxin (R & D Systems, Minneapolis, MN, USA) or RANTES (Sigma, St Louis, MO, USA) in 96well flatbottomed plates in the presence or absence of 1 ng·mL−1 interleukin (IL)‐5 or granulocyte macrophagecolony stimulating factor (GMCSF) (R & D Systems) for 15 min at 37°C. In some experiments, eosinophils were pretreated with an antiIL‐5 receptor alpha (IL5Rα) antibody (mouse IgG1κ; Pharmingen, San Diego, CA, USA) or an isotypematched control (Pharmingen), both at 0.2 µg·mL−1, for 60 min at 4°C, or a CC chemokine receptor (CCR) 3 antagonist (Compound X; a gift from Banyu Pharmaceutical Co., Ltd., Tsukuba, Japan) for 30 min at 37°C. ROS production was evoked by adding 50 µL calcium ionophore A23187 (Sigma; final concentration 1×10−5 M) to 100 µL eosinophil suspension (5×104 cells) containing 0.25 mM luminol (Futaba Medical, Tokyo, Japan). Maximal and integral intensity chemiluminescence were determined for 60 min using an ARGUS50/2D luminometer (Hamamatsu Photonics, Hamamatsu, Japan).
Flow cytometric analysis of eosinophil surface CC chemokine receptor 3
Purified eosinophils (<1×106 cells) were incubated with a fluorescein isothiocyanate (FITC)conjugated antihuman CCR3 monoclonal antibody (mouse IgG2; DAKO, Glostrup, Denmark; 0.5 µg·mL−1) for 30 min at 37°C. An FITCconjugated IgG2 isotypematched control monoclonal antibody (BecktonDickinson, San Jose, CA, USA; 0.5 µg·mL−1) was applied to assess the degree of nonspecificity. After washing the cells, thestained cells were analysed using a FACScan flow cytometer (BecktonDickinson).
Measurement of intracellular calcium concentration
Purified eosinophils from normal subjects were suspended in Hank's balanced salt solution (HBSS) containing Ca2+ (0.14 g·mL−1 CaCl2), Mg2+ (0.1 g·mL−1 MgCl2·6H2O; 0.1 g·mL−1 MgSO4·7H2O) and 2% foetal calf serum (Sigma) at a cell density of2×106 cells·mL−1. Fura‐2‐acetoxymethyl ester (DOJINDO, Kumamoto, Japan) was added at a final concentration of 2 µM. After incubation for 40 min, excess dye was removed by centrifugation for 5 min at 270×g at 4°C, and the cells were resuspended in HBSS containing 20 mM hydroxyethyl piperazine ethane sulphonic acid (HEPES) (pH 7.4) at a concentration of2×106 cells·mL−1. Calcium influx was measured using excitation at 340 and 380 nm in a fluorescence spectrometer (ARGUS; Hamamatsu Photonics).
Statistical analysis
Data were analysed using paired and unpaired t‐tests, analysis of variance (ANOVA) or the MannWhitney U‐test. A p‐value of ≤0.05 was considered to indicate significance.
Results
Luminoldependent chemiluminescence in eosinophils from normal and allergic subjects
ROS production by eosinophils was examined in terms of luminoldependent chemiluminescence evoked by calcium ionophore A23187, and compared between normal subjects and allergic patients. ROS production from eosinophils as measured via integral intensity was significantly greater in allergic patients than normal subjects (fig. 1⇓) (p<0.05).
Reactive oxygen species production by eosinophils from normal (n=12) and allergic (n=15) subjects as determined by a) maximal and b) integral intensity luminoldependent chemiluminescence for 60 min. Eosinophil stimulation was performed by adding 50μL calcium ionophore A23187 (final concentration 1×10−5 M) to 100 μL eosinophil suspension (5×104 cells) containing 0.25 mM luminol. Data are presented as mean±sem. cpm: counts per minute. *: p<0.05 versus normal subjects (unpaired t‐test).
Effect of eotaxin and regulated on activation, normal T‐cell expressed and secreted on reactive oxygen species production by eosinophils from normal and allergic subjects
The priming effect of eotaxin and RANTES on ROS production was compared in normal subjects and allergic patients. Figures 2a and b⇓ show the maximal and integral intensity. Preincubation of eosinophils with eotaxin clearly enhanced ROS production in allergic patients, but not in normal subjects. The difference between allergic and normal subjects in ROS production was much greater in the eotaxinprimed condition. In order to investigate the augmentative effect of eotaxin, results were also expressed in relation to those without chemokines (figs 2c and d⇓). The augmentative effect of eotaxin was more potent in eosinophilsfrom allergic patients than in those from normal subjects. A similar effect of RANTES was also observed (fig. 3⇓).
Reactive oxygen species (ROS) production by eosinophils treated with eotaxin (1–100 nM) from normal (□; n=12) and allergic (└; n=15) subjects as determined by maximal (a, c) and integral (b, d) intensity luminoldependent chemiluminescence for 60 min. Eosinophil stimulation was performed by adding 50 µL calcium ionophore A23187 (final concentration 1×10−5 M) to 100 µL eosinophil suspension (5×104 cells) containing 0.25 mM luminol. Data are presented as mean±sem. Preincubation of eosinophils with eotaxin enhances ROS production in allergic patients as well as normal subjects. The priming effect of eotaxin is more potent in eosinophils from allergic subjects than in those from normal subjects. cpm: counts per minute. *,**: p<0.05, p<0.01 versus control (analysis of variance); #, ##: p<0.05, p<0.01 versus normal subjects (unpaired t‐test).
Reactive oxygen species (ROS) production by eosinophils treated with regulated on activation, normal T‐cell expressed and secreted (RANTES; 1–100 nM) from normal (□; n=12) and allergic (└; n=15) subjects as determined by maximal (a, c) and integral (b, d) intensity luminoldependent chemiluminescence for 60 min. Eosinophil stimulation was performed by adding 50 µL calcium ionophore A23187 (final concentration 1×10−5 M) to 100 µL eosinophil suspension (5×104 cells) containing 0.25 mM luminol. Data are presented as mean±sem. Preincubation of eosinophils with RANTES enhances ROS production in allergic patients as well as normal subjects. The priming effect of RANTES is more potent in eosinophils from allergic subjects than in those from normal subjects. cpm: counts per minute. *,**: p<0.05, p<0.01 versus control (analysis of variance); #, ##: p<0.05, p<0.01 versus normal subjects (unpaired t‐test).
Effect of CC chemokine receptor 3 antagonist on chemokineprimed reactive oxygen species production by eosinophils
In order to confirm the involvement of CCR3 in chemokineprimed ROS production, the effect of a CCR3 antagonist thatinhibits the binding of eotaxin to human eosinophils 11 wasinvestigated. The CCR3 antagonist completely inhibited eotaxin and RANTESprimed ROS production (fig. 4⇓).
Effect of CC chemokine receptor (CCR)3 antagonist on chemokineprimed reactive oxygen species (ROS) production by eosinophils as determined by integral intensity luminoldependent chemiluminescence for 60 min (└: control; □: chemokine; ┼: chemokine plus antagonist). Purified eosinophils (1×106 cells·mL−1) obtained from allergic patients (n=5) were incubated with CCR3 antagonist (Compound X; 1×10−6 M) for 30 min. The eosinophils were then treated with eotaxin or regulated on activation, normal T‐cell expressed and secreted (RANTES) (both 100 nM) for 15 min. Eosinophil stimulation was performed by adding 50 µL calcium ionophore A23187 (final concentration 1×10−5 M) to 100 µL eosinophil suspension (5×104 cells) containing 0.25 mM luminol. Data are presented as mean±sem. CCR3 antagonist completely inhibited the priming effect of eotaxin and RANTES. cpm: counts per minute. *, **: p<0.05, p<0.01 (paired t‐test).
CC chemokine receptor 3 expression on eosinophils from normal and allergic subjects
In order to investigate the different response of eosinophils from allergic patients, expression of CCR3, a common receptor for RANTES and eotaxin, was determined. The percentage of CCR3positive cells and mean fluorescent intensity compared to controls were used as parameters of receptor expression. No significant differences in either were observed between eosinophils from allergic and normal subjects (fig. 5⇓).
Fluorescenceactivated cellsorting analysis of CC chemokine receptor (CCR)3 expression on eosinophils treated with fluorescein isothiocyanateconjugated monoclonal antibodies directed against CCR3 from a) normal subjects (n=7) and b) allergic patients (n=8) (□: immunoglobulin G2a (negative control); ▓: CCR3). Representative histograms are shown. No significant difference in surface expression was observed between eosinophils from allergic and normal subjects (14.5±2.2 versus 13.9±1.8 difference in mean fluorescence intensity (MFI) from negative control; 82.2±6.7 versus 83.0±7.8% CCR3positive cells).
Effect of interleukin‐5 on chemokineprimed reactive oxygen species production by eosinophils
IL‐5 has been shown to enhance the effector function of eosinophils 12, 13. IL‐5 augments eosinophil responses to plateletactivating factor, formylmethionylleucylphenylalanine, platelet factor‐4 and complement factor 5a 13, 14. Therefore, the possible involvement of IL‐5 in the different eosinophil responses to chemokines between normal subjectsand patients was examined. In a preliminary study, a 1‐ng·mL−1 dose of IL‐5, as indicated in previous reports of serum concentrations in allergic patients 15, had no effect onROS production by eosinophils in the absence of chemokines (fig. 6a⇓). In normal subjects, the action of IL‐5 further enhanced the priming effect of chemokines (figs 6b and c⇓). Interestingly, this effect was not observed in eosinophils from allergic patients (fig. 6d⇓).
Effect of interleukin (IL)‐5 on reactive oxygen species (ROS) production by a) control (C; normal subjects) and b) chemokineprimed (normal (N) or allergic (A) subjects) eosinophils as determined by integralintensity luminoldependent chemiluminescence for 60 min. Purified eosinophils (1×106 cells·mL−1) obtained from normal (n=7) and allergic (n=6) subjects were preincubated with eotaxin or regulated on activation, normal T‐cell expressed and secreted (RANTES) (both 10 nM) in the presence (└) or absence (□) of IL‐5 (1 ng·mL−1). Eosinophil stimulation was performed by adding 50 µL calcium ionophore A23187 (final concentration 1×10−5 M) to 100 µL eosinophil suspension (5×104 cells) containing 0.25 mM luminol. Data are presented as mean±sem. In a preliminary study, a 1‐ng·mL−1 dose of IL‐5 alone did not affect ROS production by eosinophils (a). In normal subjects, the action of IL‐5 further enhances the priming effect of chemokines. This effect is not observed in eosinophils from allergic patients. cpm: counts per minute. *, **: p<0.05, p<0.01 versus chemokine plus IL‐5 (paired t‐test).
In addition, GMCSF (1 ng·mL−1) did not affect the priming effect of eotaxin or RANTES (125.5±17.6 versus 119.7±9.4% control integral chemiluminescent intensity, eotaxin alone versus eotaxin plus GMCSF). Moreover, the CCR3 expression of eosinophils did not change after treatment with IL‐5 (fig. 7⇓).
Fluorescenceactivated cellsorting analysis of CC chemokine receptor (CCR) 3 expression on eosinophils from normal subjects (n=5) treated with fluorescein isothiocyanateconjugated monoclonal antibodies directed against CCR3 after incubation with a) phosphatebuffered saline (PBS) and b) interleukin (IL)‐5 (1 ng·mL−1) for 30 min (□: immunoglobulin G2a (negative control); ▓: CCR3). Representative histograms are shown. Eosinophil CCR3 expression did not change after the 30min treatment with IL‐5 compared to PBS (14.8±2.5 versus 15.4±1.7 difference in mean fluorescence intensity (MFI) from negative control; 85.5±5.5 versus 89.7±3.5% CCR3positive cells).
In order to investigate whether blockade of IL5Rα on allergic eosinophils is able to reverse this augmentative effect on chemokine priming, allergic eosinophils were preincubated with the antiIL5Rα antibody prior to eotaxin stimulation. Blockade of the IL‐5 receptor did not affect the priming effect of eotaxin in allergic eosinophils (156.6±10.2 versus 159.6±18.8% control integral chemiluminescent intensity, eotaxin alone versus eotaxin plus antiIL5Rα; n=4).
Effect of interleukin‐5 on chemokineinduced calcium influx in eosinophils
In order to study whether IL‐5 modulates the downstream signalling of CCR3 to enhance the response to eotaxin, the effect of IL‐5 on chemokineinduced calcium influx was investigated. However, IL‐5 did not affect the calcium influx induced by eotaxin (fig. 8⇓).
Effect of interleukin (IL)‐5 on calcium influx into eosinophils induced by eotaxin. Eosinophils obtained from normal subjects were preincubated in the a) absence and b) presence of IL‐5 (1 ng·mL−1). The eosinophils were then stimulated with eotaxin at a final concentration of 100 nM (arrow) and calcium influx was measured as described in the Measurement of intracellular calcium concentration section. The data shown are representative of three independent analyses from different donors, each showing similar results. Preincubation with IL‐5 did not affect calcium influx induced by eotaxin. RF: relative fluorescence.
Discussion
Several studies have reported that eosinophil function is highly dependent on the pathophysiological conditions of allergic disease 7, 16–19. The present study shows upregulated oxidative metabolism in eosinophils obtained from allergic patients compared to those from normal subjects. Asimilar increase in ROS production by eosinophils was observed in allergic patients 16, 17. It has also been demonstrated that eosinophils from subjects undergoing allergen challenge or patients with such symptoms exhibit enhanced ROS production 18, 19. Taking the results of these studies together with the present observations, eosinophils from allergic patients may have already been activated in the peripheral blood stream before they infiltrate the tissues.
Moreover, in the present study, functional upregulation of the response to chemokines in ROS production by eosinophils obtained from allergic patients was observed. The priming effect of both RANTES and eotaxin on ROS production was significantly greater than that on eosinophils from normal subjects. Even at the suboptimal dose for eosinophils from normal subjects, eosinophils from allergic patients showed enhanced ROS production after treatment with chemokines. These results suggest that eosinophils from allergic patients are more sensitive and responsive to chemokines.
It has been reported that eotaxin and IL‐5 cooperate to regulate eosinophil trafficking during allergic inflammation 20, 21. Schweizer et al. 22 reported that chemokineinduced responses are very sensitive to priming by cytokines such as IL‐5. Therefore, in order to extend understanding of these upregulated sensitivities and their responsiveness to chemokines, the possible involvement of cytokines, such as IL‐5 and GMCSF, in the priming effect of chemokines was examined. It was demonstrated that a low concentration (1 ng·mL−1) of IL‐5 enhanced chemokineprimed ROS production by eosinophils, suggesting that IL‐5 may enhance the responsiveness to chemokines. Although a similar tendency has been observed in other eosinophil functions, such as degranulation and migration 14, 22, 23, this is the first report of a priming effect of IL‐5 on chemokineprimed ROS production from eosinophils. Interestingly, no augmentative effect of GMCSF was demonstrated despite the β subunit (βc) being common to both IL‐5 and GMCSF receptors. Although βc plays a major role in IL‐5 signalling 24, recent evidence indicates that the specific IL‐5 receptor IL‐5Rα is also involved in signal transduction. Geijsen et al. 25 have cloned an IL5Rαassociated molecule, syntenin, which is required for activation of the transcription factor Sox4. IL5Rα also associates with a novel signalling molecule, IL‐5 receptorinteracting protein, which activates Lyn and Hck in eosinophils 26. Therefore, these IL5Rαspecific molecules may be responsible for the distinct response to IL‐5.
It has been reported that IL‐5 is produced by eosinophils themselves, especially in allergic conditions 27. One possibility is that allergic eosinophils can be primed by IL‐5 produced by themselves. However, it was demonstrated thatblockade of the IL‐5 receptor on allergic eosinophils could not reverse the priming effect of chemokines. This resultindicates that the upregulated response to chemokines observed in allergic eosinophils was not elicited by IL‐5 produced after eosinophil isolation. Moreover, the augmentative effect of IL‐5 was observed only in eosinophils from normal subjects and not in those from allergic patients. This distinct phenotype is in line with data demonstrating in vivo priming of adhesionassociated responses of peripheral blood eosinophils of patients with allergic diseases 13, 14. Therefore, eosinophils from allergic patients may undergo IL‐5 exposure in the blood stream, resulting in great enhancement of responsiveness to chemokines, as demonstrated in the present study.
The possibility of differences in expression of CCR3 as a means of explaining the different responses to chemokines was examined, but no significant difference was found in CCR3 expression between patients and normal subjects. Furthermore, CCR3 expression of eosinophils did not change after treatment with IL‐5. These observations suggest that functional upregulation of response through CCR3 in allergic patients does not depend on an increase in CCR3 expression. As regards signalling of eosinophils, it was examined whether IL‐5 modulates the calcium mobilisation induced by chemokines. However, IL‐5 did not affect the intracellular calcium influx induced by eotaxin. It has recently been reported that the baseline activity of phosphatidylinositol 3‐kinase is elevated in allergic patients compared to normal subjects, together with involvement of IL‐5 in phosphatidylinositol 3‐kinase activation 28, 29. Thus, it may be assumed that IL‐5 modulates the downstream signalling of CCR3 to enhance the response to eotaxin. Beside the involvement of cytokines, such as IL‐5, in the upregulated response of eosinophils from allergic patients to chemokines, it can be presumed that other mechanisms, such as CCR3 polymorphism 30 and change in affinity/avidity, are involved. Recently, CCR3 has become a target in the treatment of allergic diseases such as asthma, atopic dermatitis and allergic rhinitis. Indeed, an inhibitory effect of CCR3 antagonist on chemokinemediated eosinophil function has been found (manuscript in preparation).
In conclusion, the present study has demonstrated an enhanced response to chemokines in the reactive oxygen species production of eosinophils from allergic patients, with the possible involvement of interleukin‐5 in that enhancement, and without changes in CC chemokine receptor 3 expression. Further studies are required to elucidate the mechanisms of the different responses of CC chemokine receptor 3.
Acknowledgments
The authors would like to thank Y. Kamada, T. Takahashi and H. Oyamada fortheir help and support.
- Received April 4, 2002.
- Accepted January 14, 2003.
- © ERS Journals Ltd
References
