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(Journal of Leukocyte Biology. 2000;68:655-661.)
© 2000 by Society for Leukocyte Biology

In vivo priming of Fc{alpha}R functioning on eosinophils of allergic asthmatics

Madelon Bracke, Ed van de Graaf, Jan-Willem J Lammers, Paul J Coffer and Leo Koenderman

Department of Pulmonary Diseases, University Medical Center, Utrecht, The Netherlands

Correspondence: L. Koenderman, Ph.D., Department of Pulmonary Diseases, Room F02.333, University Medical Center, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. E-mail: L.Koenderman{at}hli.azu.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inflammation in allergic asthma is characterized by an influx of eosinophils and the presence of eosinophil products in the bronchial tissue. Orchestration of this inflammatory response is in part mediated by cytokines and chemoattractants, but final activation can require additional stimuli. IgA, the most abundant immunoglobulin at mucosal surfaces, is potentially a potent trigger for eosinophil activation. Previously, we have shown that binding IgA-coated targets is dependent on in vitro stimulation of cells with cytokines. Here, we demonstrate that eosinophils isolated from the blood of allergic asthmatic patients bind IgA beads independently of prior in vitro stimulation. Furthermore, we found that the proinflammatory cytokine, TNF-{alpha}, is a potent enhancer of IgA binding to eosinophils from allergic asthmatics, and it does not activate Fc{alpha}R on eosinophils isolated from normal donors. The difference in IgA binding by Fc{alpha}Rs on normal and patient eosinophils might be explained by the activation of different signal transduction pathways. Studying intracellular signaling, we found an enhanced basal activity of phosphatidylinositol 3-kinase (PI3K) in eosinophils derived from allergic asthmatics. Moreover, inhibition of PI3K in these cells blocked the background and the TNF-{alpha}-induced IgA binding completely. In summary, these data demonstrate that the responsiveness of human eosinophils to TNF-{alpha} might be an important contribution for fine-tuning the allergic inflammatory reaction. Furthermore, the preactivation of PI3K results in a broader sensitivity to subsequent challenge with inflammatory cytokines.

Key Words: FC{alpha}R • TNF • signal transduction • allergic asthma • PI3K


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Eosinophils play an important role in allergic disorders such as allergic asthma [1 ]. Upon allergen challenge, eosinophils migrate from the peripheral blood into allergic inflammatory tissues and are subsequently activated. In bronchoalveolar lavage fluid obtained from asthmatic patients, toxic granule products, such as eosinophil cationic protein and major basic protein, can be detected [2 ]. The release of these toxic eosinophil proteins (degranulation) can result in damage of the respiratory epithelium, leading to airway hyperresponsiveness [3 , 4 ]. Also, production of toxic oxygen metabolites during the so-called respiratory burst [5 ] and synthesis and secretion of bioactive lipid mediators can contribute to airway hyperreactivity [6 , 7 ]. Despite these clear indications of activation, relatively little is known about the processes that lead to activation of eosinophils in vivo.

Several mechanisms are involved in eosinophil activation, including activation via adhesion molecules and receptors for complement factors and immunoglobulins [8 ]. These receptors are potent signaling molecules in vitro, albeit optimal only after priming with cytokines or chemotaxins [9 , 10 ]. Binding of cytokines to specific transmembrane receptors results in intracellular increases in tyrosine phosphorylation and activation of distinct signal transduction pathways [11 ], leading finally to activation of effector functions of target cells. Therefore, preactivation of eosinophils with cytokines is a critical step in the process of activation. The influence of priming on the functioning of immunoglobulin (Ig)A and IgG receptors is thought to be important, because Igs are likely to be involved in the activation processes of eosinophils in vivo. The receptor for IgA is a possible candidate for final eosinophil activation at allergic inflammatory sites, because IgA is present abundantly on mucosal surfaces, and IgA-coated surfaces potently induce eosinophil degranulation [12 13 14 ].

We have demonstrated previously that activation of the Fc receptor(s) on eosinophils from healthy donors is regulated by Th2-derived cytokines, such as interleukin (IL)-4 and IL-5 [15 16 17 ]. In the present study, we show that when eosinophils are derived from the blood of allergic asthmatics, IgA binding is possible without prior in vitro priming with cytokines. Although eosinophils isolated from normal donors do not respond to stimulation with tumor necrosis factor {alpha} (TNF-{alpha}), eosinophils of allergic patients show an enhanced IgA binding capacity after TNF-{alpha} stimulation. To investigate the priming state and the effect of TNF-{alpha} on a molecular level, we have studied the cytokine-induced activation of phosphatidylinositol 3-OH kinase (PI3K) and p38 mitogen-activated protein kinases (MAPK) in freshly isolated eosinophils from normal donors and allergic asthmatics. Pharmacological inhibitors of these kinases were used to study the involvement of specific signaling pathways in the priming state of the eosinophils and their role in the activation of the receptor for the Fc portion of IgA (Fc{alpha}R).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents: cytokines, antibodies, and pharmacological inhibitors
Ficoll-paque, Percoll, and protein A-sepharose were obtained from Pharmacia (Uppsala, Sweden). Ovalbumin were purchased from Sigma Chemicals (St. Louis, MO). Human serum albumin (HSA) was from the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (Amsterdam, the Netherlands). Purified human serum IgA (20 mg/ml) was obtained from Cappel (Malvern, PA); it contained no detectable trace of IgG, IgM, or non-Ig serum proteins. Recombinant human IL-5 was a gift from Dr. M. McKinnon, GlaxoWellcome (Stevenage, UK). IL-4 was purchased from Genzyme (Leuven, Belgium) and TNF-{alpha} (108 IU/mg protein), from Boehringer Mannheim (Mannheim, Germany). Other materials were reagent grade. Experiments were performed in incubation buffer [20 mM HEPES, 132 mM NaCl, 6.0 mM KCl, 1.0 mM MgSO4, 1.2 mM KH2PO4, supplemented with 5 mM glucose, 1.0 mM CaCl2, and 0.5% (w/v) HSA]. Antibodies used were: monoclonal anti-human TNF-RI and TNF-RII (mouse IgG1 and mouse IgG2a, respectively), purchased from R&D Systems (Minneapolis, MN); and antiphosphotyrosine polyclonal PKB/Akt (Ser473) and p38 MAPK (Thr180/182), from New England Biolabs (Berverly, MA). Pharmacological inhibitors LY294002 and SB203580 were purchased form BioMol (Plymouth Meeting, PA).

Patients
For this study, patients were included according to the criteria of the American Thoracic Society [18 ]. Fourteen patients (four male) with an allergic bronchial asthma were studied. The median age was 34 (range, 17–44). Patients had respiratory complaints and were examined for asthmatic symptoms. All patients had a history of bronchial hyperreactivity. The median FEV1 was 78.5% of predicted (range, 56%–131%). Patients had a reversibility of the FEV1 of more than 9% of the predicted value upon ß-2-agonist (salbutamol). Five patients were on inhaled steroids, but the data were not distinguishable from the data obtained with the cells of the steroid-naïve patients. All patients had documented allergy to one or more inhalation allergens, including house dust mite, pollen, and cat allergens, as shown by positive skin-prick tests or RAST. The study was approved by the hospital ethics committee, and all patients gave informed consent before entering into it.

Isolation of eosinophils
Blood was obtained from healthy volunteers from the Red Cross Blood Bank (Utrecht, The Netherlands) and from allergic asthmatic patients. Granulocytes from healthy volunteers were isolated from the buffy coat of 500 ml anticoagulated blood with 0.4% (w/v) trisodium citrate (pH 7.4), as described previously [19 ]. Mononuclear cells were removed by centrifugation over isotonic Percoll (1.078 g/ml). After lysis of the erythrocytes with an ice-cold NH4Cl solution, the eosinophils were isolated by the method described by Hansel et al. [20 ]. Briefly, the granulocytes were washed and resuspended in RPMI 1640 (Gibco, Paisley, UK) with 0.5% (w/v) HSA. Granulocytes were incubated for 30 min at 37°C to restore the initial density of the cells. Thereafter, the cells were washed and resuspended in phosphate-buffered saline (PBS) supplemented with 0.5% HSA and 13 mM trisodium citrate. Subsequently, an enriched population of eosinophils was obtained by centrifugation (20 min, 1000 g) over isotonic Percoll (density 1.084 g/ml, layered on Percoll with a density of 1.1 g/ml), washed, and resuspended in cold PBS. Cells were incubated with a CD16 monoclonal antibody (mAb; 5D2; 2.5 µg/107 cells) during 20 min at 4°C. Subsequently, anti-mouse IgG immunomagnetic beads were added, and neutrophils were removed by a magnetic particle concentrator (MCP-Dynal). Purity of eosinophils was always >95%, and recovery was 60%–70%.

From allergic asthmatic patients, 30 ml blood was collected via venipuncture and was anticoagulated with sodium heparine. Mononuclear cells were removed by centrifugation over isotonic Percoll (1.078 g/ml). After lysis of the erythrocytes with an ice-cold NH4Cl solution, the eosinophils were isolated as described above. Purity of eosinophils was always >90%, and recovery was usually 3–6 x 106 cells.

IgA binding assays
IgA binding assays were performed with purified human eosinophils from normal donors or allergic asthmatics. Before performing a binding assay, purified eosinophils were washed with Ca2+-free incubation buffer containing 0.5 mM EGTA and brought to a concentration of 8 x 106 cells/ml. Cell suspension (50 µl; 0.4x106 cells) was incubated at 37°C, with or without cytokines. Cells were stimulated with IL-4 (10-9 M; 5 min), IL-5 (10-9 M; 15 min), or TNF-{alpha} (102 U/ml; 15 min). After stimulation of the cells, dynabeads coated with serum IgA (10 mg/ml), as described previously [15 ], were added at a ratio of 3.5 beads/cell. After briefly mixing, the cells and beads were pelleted for 15 sec at 100 rpm and incubated 20 min (IL-4-treated cells) or 30 min (IL-5- and TNF-{alpha}-treated cells) at 37°C. Afterward, incubation cells were resuspended vigorously, and IgA binding was evaluated under a microscope. All cells that had bound two beads or more were defined as rosettes. One hundred cells were scored, and the number of beads that was bound to the cells was counted. The amount of beads bound to a total of 100 cells (bound and unbound to beads) was designated as the rosette index. For inhibition studies, cells were preincubated with specific inhibitors before stimulation with cytokines. Cells were incubated with the PI3K inhibitor, LY294002, or the p38 inhibitor, SB203580, for 15 min at a final concentration of 1 µM.

Detection of TNF-{alpha} receptors on human eosinophils
TNF-{alpha} receptors on human eosinophils were detected using mouse mAbs directed against the human TNF-RI and TNF-RII and a secondary fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse antibody in a flowcytometric assay (FACS Vantage flow cytometer, Becton Dickinson, San Jose, CA). D6 (IgG1), a mAb against a bacterial epitope, was used as a negative control.

PKB and p38 MAPK phosphorylation
Eosinophils were isolated as described above and incubated at 37°C for 30 min in incubation buffer. For detection of phosphorylation of PKB or p38 MAPK, eosinophils (0.5x106 per condition) were washed twice in ice-cold PBS after stimulation with cytokines and lysed in lysis buffer (1% Triton X-100, 50 mM Tris-Cl, pH 8.0, 100 mM NaCl) with phosphatase inhibitors. Subsequently, 5x Laemmli sample buffer was added, and the lysates were boiled for 5 min. Total cell lysates were analyzed on 15% sodium dodecyl sulfate (SDS)-polyacrylamide gels. Proteins were transferred to Immobilon-P and incubated with blocking buffer [Tris-buffered saline/Tween 20 (TBST) supplemented with 1 mM ethylenediaminetetraacetate (EDTA) and 0.6% bovine serum albumin (BSA)] with polyclonal phospho-PKB (Ser473) or phospho-p38 MAPK (Thr180/182) antisera. Detection was with enhanced chemiluminescence (ECL; Amersham, UK). Data on the autoradiograms were analyzed densitometrically (Molecular Dynamics, Sunnyvale, CA), using ImageQuant software. Data are presented as arbitrary densitometric counts.

Statistical analysis
Results of rosette experiments were expressed as means ± SE. Statistical analysis was performed by using paired and unpaired Student’s t-tests. In all experiments, P values < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Difference in IgA binding to eosinophils of healthy donors and allergic asthmatics
To investigate a possible role for Fc{alpha}R in the chronic inflammatory process of allergic asthma, we compared the ability of eosinophils isolated from the blood of healthy individuals with that of allergic asthmatics to bind IgA-coated particles. As we have described previously, eosinophils from normal donors did not bind IgA beads unless they were pretreated with cytokines, such as IL-4 or IL-5 [15 ]. In contrast, freshly isolated eosinophils of allergic asthmatics showed a significant increase in IgA binding without preincubation with cytokines (Fig. 1 ). This enhanced IgA binding suggests that eosinophils in the blood of allergic asthmatics were already in a "primed" state in vivo.



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  		Figure 1. IgA binding to eosinophils from healthy and asthmatic individuals. IgA binding assays were performed with freshly isolated eosinophils from normal donors (control; left) or allergic asthmatics (patients; right). Binding of IgA beads to these cells was measured, and results are expressed as rosette index (number of beads/100 cells). Each symbol indicates an individual donor, and the means are indicated as a line (—). The number of donors in each group is indicated between brackets. Values between the two groups of donors differed significantly (p<0.05), indicated with *.

 
To further define this preactivated state in the context of IgA binding, we investigated whether the binding of IgA-coated particles to eosinophils of asthmatics could be increased by incubation with Th2 cytokines. Indeed, stimulation of the cells with IL-4 or IL-5 increased the IgA binding to patient eosinophils, similarly to normal eosinophils (Fig. 2 ). Surprisingly, IgA binding was not modulated by TNF-{alpha} on eosinophils isolated from normal donors, although cells isolated from allergic patients showed a clear response to TNF-{alpha} stimulation (Fig. 2) .



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  		Figure 2. Effect of priming on the binding to eosinophils from healthy and asthmatic individuals. The effect of cytokine stimulation on the binding of IgA-coated beads to eosinophils from normal donors (open bars) and allergic asthmatics (solid bars) was compared. Prior to performing rosette assays (see Materials and Methods), cells were preincubated with IL-4, IL-5, or TNF-{alpha}. Results are expressed as rosette index (number of beads/100 cells) and as means ± SE (n=8). A statistical difference (p<0.01) was present among rosette formation of patient cells in the presence and absence of TNF-{alpha} (see also Fig. 5 ). Values indicated with * differed significantly (p<0.05) between the two groups of donors.

 
Detection of TNF-{alpha} receptors on human eosinophils of normal donors and allergic asthmatics
To explain the difference in TNF-{alpha} response of eosinophils from normal donors and patients, first we investigated the levels of expression of the receptors for TNF-{alpha} on the cells. Expression of two TNF-{alpha} receptors was detected on human eosinophils in a flowcytometric assay using mAbs against the anti-human TNF-RI (55 kDa) and TNF-RII (75 kDa). As shown in Figure 3A , similar levels of expression for both receptors were detected on the surface of unstimulated human eosinophils isolated from the blood of normal donors (left panel) or allergic asthmatics (right panel). Incubation with cytokines did not enhance the expression of the receptors (unpublished results). For comparison, the expression levels of both receptors are shown in Figure 3B . Because receptors for TNF-{alpha} are present not only on eosinophils of allergic patients but also on eosinophils of nonallergic donors, this does not explain the difference in TNF responsiveness of the cells. Therefore, we investigated whether the TNF receptors present were functional in the context of signal transduction.



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  		Figure 3. FACS analysis of TNF-R expression on eosinophils from normal donors (left panel) and allergic asthmatics (right panel). Eosinophils derived from normal individuals and from allergic patients were stained with mAbs against human TNF-RI and TNF-RII. D6 (IgG1), a mAb against a bacterial epitope, was used as a negative control. Data obtained with neutrophils are shown in B. The experiment shown is representative of four additional experiments, and the same expression was observed for primed and unprimed cells.

 
Activation of PI3K-PKB/c-Akt pathway by TNF-{alpha} in human eosinophils
We investigated the signal transduction pathways that were demonstrated to be important for the modulation of FC{alpha}R by cytokines [16 ]. Because we have demonstrated previously a critical role for PI3K activity in cytokine-induced activation of FC{alpha}R on human eosinophils [16 ], we investigated first whether TNF-{alpha} could activate this lipid kinase. In vitro immune-complex kinase assays are commonly performed to measure the activity of PI3K. However, the sensitivity of this assay is dependent on large amounts of cells (>107/sample), which could not be isolated from the small volume of blood obtained from allergic patients. Therefore, we used an indirect method to investigate PI3K activity. Previously, it has been described that activation of PI3K results in the activation (by phosphorylation) of PKB/c-Akt, a serine/threonine protein kinase [21 ]. In this way, we utilized the phosphorylation of PKB as a measure for PI3K activation in eosinophils of normal individuals and allergic asthmatics. We studied the effect of TNF-{alpha} and IL-5 stimulation on the phosphorylation of PKB, which can be detected by western blotting with a phospho-specific (Ser473) anti-PKB antibody. In eosinophils freshly isolated from the blood of healthy individuals, no phosphorylation of PKB was observed as shown in Figure 4A . This was not enhanced by incubation of these cells with TNF-{alpha} (100 U/ml). However, phosphorylated PKB was clearly present after incubation of the cells with IL-5 (5x10-10 M), indicating that PI3K can be activated upon cytokine stimulation. In untreated eosinophils from allergic asthmatic patients, PKB was present already in a phosphorylated form (Fig. 4A , lane 4), which is only modestly increased by cytokine stimulation. Densitometric analysis of the four different experiments corroborates these findings (see Fig. 4B ).



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  		Figure 4. The effect of TNF-{alpha} on the phosphorylation of PKB (A) and p38 MAPK (B) in human eosinophils from normal donors and asthmatic patients. Eosinophils were isolated from the blood of a normal donor and an allergic asthmatic. After stimulation of the cells with either buffer (-), TNF-{alpha} (100 U/ml), or IL-5 (10-9 M) for 10 min at 37°C, eosinophils (0.5x106 per condition) were washed with ice-cold PBS, lysed in lysis buffer, and heated for 5 min after addition of 5x sample buffer. Phosphorylation of PKB (A) was detected using a polyclonal antiphospho-PKB antiserum for western blotting, and an antiphospho-p38 MAPK was used to detect phosphorylated p38 MAPK (B). The blots shown are representative for four experiments. Densitometrical corroboration of the data is shown in C. These results are expressed as means of the data obtained from four different experiments.

 
Activation of p38 MAPK by TNF-{alpha} in human eosinophils
In addition to activation of PI3K in human eosinophils, we described previously the activation of p38 MAPK by Th2 cytokines, IL-4 and IL-5 [16 ]. Also previously, it has been described that TNF-{alpha} can activate p38 MAPK in neutrophils [22 , 23 ]; however, whether TNF-{alpha} can also activate this pathway in human eosinophils has not been described. We, therefore, investigated the activation of p38 MAPK by TNF-{alpha} in eosinophils derived from the blood of allergic and normal donors. As is shown in Figure 4B , stimulation of human eosinophils with TNF-{alpha} (100 U/ml) for 10 min resulted in phosphorylation of p38 MAPK, as detected by using a specific antiphospho-p38 MAPK antibody. In contrast with the difference in basal levels of PKB phosphorylation (Fig. 4A) , the levels of p38 MAPK phosphorylation in unstimulated eosinophils were comparable between normal individuals and allergic asthmatics (Fig. 4B) . Thus, this suggests that the activation state of p38 MAPK or ERK2 is not likely to be a crucial step in mediating TNF-{alpha} responsiveness of eosinophils from allergic patients.

The PI3K-inhibitor, LY294002, inhibits IgA-rosette formation to eosinophils of allergic asthmatics
To investigate the importance of PI3K and p38 MAPK in the activation of Fc{alpha}R on eosinophils of asthmatic patients compared with normal donors, we studied the effect of pharmacological inhibitors on IgA binding. As shown in Figure 5 , TNF-{alpha}- and IL-5-induced binding of IgA beads to patient eosinophils was blocked by pretreatment with p38 MAPK inhibitor, SB203580. Inhibition of p38 MAPK activity reduced the cytokine-stimulated IgA binding, however cells are still able to bind IgA, suggesting that these cells are still primed. In contrast, inhibition of PI3K activity by pretreatment with LY294002 resulted in a complete abrogation of IgA binding. LY294002 also prohibited the binding of IgA beads to unstimulated eosinophils isolated from allergic asthmatics. This thus suggests that the in vivo priming status of eosinophils in the peripheral blood of allergic asthmatics is (reversibly) mediated by preactivation of PI3K.



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  		Figure 5. Effect of inhibiting PI3K and p38 MAPK on IgA binding to cytokine-stimulated eosinophils from normal donors and allergic asthmatic patients. Cells were pretreated for 15 min at 37°C in the absence (open bars) or presence of 1 µM LY294002 (shaded bars) or 1 µM SB203580 (solid bars), and subsequently stimulated at 37°C with TNF-{alpha} (100 U/ml) or IL-5 (10-9 M) for 15 min. Subsequently, cells were incubated with IgA beads for 30 min. Binding IgA beads to the cells is indicated as rosette index (number of beads/100 cells). Mean values are presented ± SE (n=3). *p<0.05; **p<0.025; ***p<0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although activation of immunoglobulin and complement receptors might be important for the final activation of eosinophils, it only occurs when the cells are primed (preactivated) with cytokines. Therefore, it is a clinically relevant finding that freshly purified eosinophils of allergic asthmatics show an enhanced IgA binding without being pretreated with cytokines (Fig. 1) . This demonstrates the in vivo priming of this cell type in these patients. Furthermore, this "primed" phenotype is in concordance with data describing in vivo priming of adhesion-associated responses of eosinophils isolated from the peripheral blood of patients with allergic diseases [24 , 25 ]. Enhanced responses, such as increased migration and enhanced respiratory burst, of eosinophils from atopic donors have been ascribed to the actions of cytokines released by Th2 lymphocytes, such as IL-5 and IL-4 [25 26 27 ]. These findings are consistent with the hypothesis that part of the priming processes occurring in vivo is of an irreversible nature. Moreover, it is very likely that not all eosinophils in blood of allergic asthmatics exhibit a similar priming state, which will result in a heterogeneous cell population in the context of cellular priming (see below).

The aforementioned cytokines can modulate eosinophil functions by a direct effect on eosinophils or indirectly by influencing bystander cells such as endothelial and epithelial cells [28 , 29 ]. It has been suggested that stimulation by IL-5/IL-3/granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokines can result in a broad range of priming responses, and IL-4 priming results in a rather restricted phenotype. It needs to be mentioned that fully primed eosinophils in vivo will extravasate rapidly into the tissue most likely and, therefore, will not be found in the blood. In contrast, eosinophils in the peripheral blood will probably show a whole range of intermediate-primed phenotypes. This explains the finding that IgA binding to eosinophils isolated from the blood of allergic donors can be further activated in vitro by cytokines (Fig. 1) . The priming of binding of IgA-coated beads to eosinophils is not mediated by an increase in receptor expression but rather an affinity/avidity switch of already expressed receptors controlled by inside-out signaling [15 , 16 , 30 ].

Apart from IL-5 and IL-4, other proinflammatory cytokines such as TNF-{alpha} are produced in the allergic inflammatory reaction [31 ]. TNF-{alpha} can be released by activated T cells (including T helper 2 cells), mast cells, macrophages, and eosinophils, and its release is enhanced upon allergen challenge [32 33 34 ]. TNF-{alpha} is a potent activator of endothelial cells and induces expression of adhesion molecules, such as intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1, which may be involved in extravasation of eosinophils into the lung tissue [35 , 36 ]. Moreover, in eosinophils, TNF-{alpha} has been shown to 1) induce the production of reactive oxygen metabolites [37 ], 2) stimulate eosinophil toxicity toward endothelium [38 ], and 3) enhance the production of leukotriene C4 in response to formyl-Met-Leu-Phe (fMLP) [39 ]. Furthermore, in bronchoalveolar lavage (BAL) fluid from allergic asthmatics, high levels of TNF-{alpha} have been detected [40 ], and late responses to allergen were found to be associated with increased concentrations of TNF-{alpha} and IL-5 in sputum [41 ]. It has been shown that TNF-{alpha}, in combination with IL-5 or IFN{gamma}, induced ICAM-1 expression on human eosinophils [42 ]. These findings indicate a role for TNF-{alpha} in the pathogenesis of allergic asthma. This has also been suggested from animal studies, because TNF-{alpha} inhalation caused increased bronchial reactivity in rats [43 ].

However, in contrast to cytokines such as GM-CSF/IL-3/IL-5, for example, relatively little is known about the mechanism by which TNF-{alpha} affects eosinophil effector functions. In human cells, two receptor structures for TNF-{alpha} (TNF-R) have been cloned [44 ]: TNF-RI (55 kDa), which mediates the majority of TNF effects, and TNF-RII (75 kDa), both belonging to the TNF-R superfamily that includes the low-affinity nerve growth factor receptor and the Fas receptor (APO-1, CD95) [45 ]. Both receptors are expressed on human eosinophils [37 ]. In this study, we show that eosinophils isolated from allergic patients are responsive to TNF-{alpha} stimulation, although eosinophils of normal donors do not react to TNF-{alpha} with an increased IgA binding (Fig. 2) . Because the expression levels of TNF-RI and TNF-RII are comparable on eosinophils from normal and allergic individuals (Fig. 3) , we suggest that the difference in responsiveness is dependent on the activation of downstream targets of the TNF-R. However, we did not find a correlation between the rosette formation of unprimed cells (Figs. 1 and 2) and the TNF-{alpha} responsiveness (Fig. 5) . Therefore, the mere responsiveness of patient cells to IgA-coated beads does not reflect the responsiveness for TNF-{alpha}.

We have described previously that IL-4, comparable with IL-5, can activate PI3K in human eosinophils [46 ]. Here, we used phosphorylation of PKB/c-akt, a serine-threonine protein kinase described as a downstream target for PI3K [21 ], as a measure for PI3K activation by TNF-{alpha}. We show that PKB phosphorylation is only slightly increased upon stimulation with TNF-{alpha} in eosinophils derived from allergic patients (Fig. 4A) . PKB phosphorylation by TNF-{alpha} in normal eosinophils, however, was not observed (Fig. 4A) .

In contrast with PI3K, p38 MAPK is activated in eosinophils of normal donors and allergic asthmatics by TNF-{alpha} (Fig. 4B) . Inhibition of p38 MAPK activity in patient eosinophils only blocks in vitro-induced IgA binding. Incubation with PI3K inhibitor, LY294002, however, results in complete abolition of the in vivo-primed IgA binding to eosinophils of allergic asthmatics (Fig. 5) . This is the first example of in vivo priming of a specific signaling pathway, and it indicates that activation of PI3K is essential for further priming by cytokines, such as TNF-{alpha}.

In Figure 6 , we present a model for the hypothesis that in vivo PI3K activation is critical for the Fc{alpha}R stimulation by TNF-{alpha}. This model predicts that PI3K and p38 MAPK need to be activated to activate ligand binding to Fc{alpha}R. In eosinophils derived from healthy donors (Fig. 6A) , in vitro stimulation with IL-4 or IL-5 results in a PI3K-mediated activation of p38 MAPK, which subsequently results in enhanced IgA binding. TNF-{alpha} treatment of these cells, however, only induces p38 MAPK activity, independently of PI3K activation. Apparently, p38 MAPK activation alone is not sufficient to activate Fc{alpha}R to bind ligand. In normal eosinophils, activation of PI3K and p38 MAPK is required to induce Fc{alpha}R activation. In freshly isolated eosinophils derived from allergic asthmatics, a certain level of PI3K activation is already present, as measured by background levels of PKB phosphorylation (Fig. 4A) . We speculate that this "in vivo" level of PI3K activity is necessary to synergize with in vitro TNF-{alpha}-induced p38 MAPK activation (Fig. 6B) . Increased binding of IgA beads to the eosinophils by TNF-{alpha} treatment can thus be explained by the activated state of the PI3K pathway in the "in vivo primed" eosinophils of asthmatics. However, we do not exclude that, except for activation of PI3K, additional signaling pathways are involved in the priming status of eosinophils. It is tempting to speculate that cytokine-mediated priming of eosinophil responses is a gradual process that can include many intermediate states of priming. Enhanced levels of priming cytokines in the peripheral blood of asthmatic patients, but also the local production in inflamed airways, might contribute to the final activation state of eosinophils. Therefore, it is extremely important that the activation state of important inflammatory receptors, such as Fc{alpha}R, is tightly regulated by the appropriate cytokines for the final effector functions of the eosinophils. Understanding the functioning of cytokines and their receptors might provide novel therapeutic options, such as targeting "primed" signaling molecules.



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  		Figure 6. Activation of PI3K determines the capacity of cytokines to induce IgA binding. Activation of PI3K and p38 MAPK is necessary for IgA binding to human eosinophils. Although IL-4 and IL-5 activate p38 in a PI3K-dependent manner, TNF-{alpha} activation of p38 MAPK occurs independently of PI3K. Because of in vivo priming, a basal activity of PI3K is present in eosinophils derived from allergic asthmatic patients (indicated in B). As a consequence, in vitro TNF-{alpha} stimulation of eosinophils isolated from allergic individuals leads to increased IgA binding through further activation of p38 MAPK. In contrast, for TNF-{alpha}-induced IgA binding to eosinophils from healthy individuals, an additional activation of PI3K would be required (A). In this model, cytokines only indicate in vitro stimulation. Thick arrows indicate the pathways that are required to obtain FC{alpha}R activation. Disruption of one of these pathways (either directly or by blocking an upstream pathway) results in inhibition of IgA binding.

 


   ACKNOWLEDGEMENTS
 
M. B. was supported by a research grant of the Dutch Asthma Foundation (NAF 94.44).


   FOOTNOTES
 
Current address of Madelon Bracke: Leukocyte Adhesion Laboratory, Imperial Cancer Research Fund, London, United Kingdom.

Received November 23, 1999; revised May 25, 2000; accepted May 26, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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