Analysis of Signal Transduction Pathways Regulating Cytokine-Mediated Fc Receptor Activation on Human Eosinophils¹

Eosinophilic granulocytes play an important role in host defense against parasites, and the regulation of their activation through the interaction with cytokines and chemoattractants is critical in regulating their functioning. Eosinophils are also implicated in the pathogenesis of a variety of inflammatory diseases which are associated with tissue damage, for example allergic asthma (1, 2). Although the exact role of eosinophils in allergic inflammation remains to be elucidated, increased numbers are regularly observed in peripheral blood of patients with allergic asthma. Furthermore, in these individuals, eosinophils exhibit a preactivated or “primed” state, indicated by an increased responsiveness toward distinct stimuli (3, 4). In addition, eosinophil-derived products are found in lung tissue, such as active eosinophil cationic protein and major basic protein. The release of such toxic mediators results in destruction and damage of the respiratory epithelial tissue and can lead to airway hyperresponsiveness (5, 6). Also, production of toxic oxygen metabolites during the so-called respiratory burst and synthesis and secretion of bioactive lipid mediators contribute to airway hyperreactivity (7, 8, 9).

Despite much knowledge concerning the effector functions of eosinophils in vivo, relatively little is known about the processes that lead to activation of eosinophils in the bronchial compartment. Several mechanisms are involved in these processes, including activation via receptors for adhesion molecules, complement factors, and Igs (10). These receptors are potent signaling molecules in vitro, albeit optimal only after priming with cytokines or chemotaxins (11, 12).

Binding of cytokines to specific transmembrane receptors results in intracellular increases in tyrosine phosphorylation and activation of distinct signal transduction pathways (13). In vitro, priming of eosinophils with IL-5 potentates effector functions such as respiratory burst activation, migration, and platelet-activating factor release (14, 15, 16, 17). Although compared with IL-5 little is known about effects of IL-4 priming on eosinophil function, it can also serve as a priming agent for eosinophil chemotaxis (18). Furthermore, functioning of IgA Ig receptors on eosinophils is strongly modulated by both IL-5 and IL-4 (19), albeit with different activation kinetics.

The receptor for Ig A is a possible candidate for final eosinophil activation at allergic inflammatory sites, since IgA is abundantly present on mucosal surfaces and IgA-coated particles potently induce eosinophil degranulation (20, 21, 22). Receptors for IgA are described on many cell types, including monocytes, neutrophils, and eosinophils (23, 24, 25). At present not much is known about the activation and functioning of FcαRs on eosinophils in contrast with the better defined FcγRII (26). Binding of IgG-coated magnetic beads or IgG-coated erythrocytes to eosinophils is sensitive for priming by IL-5 and granulocyte-macrophage CSF, but not influenced by priming with IL-4 (19, 27).

To investigate which signal transduction pathways are involved in the regulation of Fc receptors on human eosinophils, we have studied the activation of phosphatidylinositol 3-OH kinase (PI3K),³ and mitogen-activated protein kinases (MAPK); i.e., extracellular regulated kinase (ERK)-1, ERK2, and p38 kinase, in freshly isolated eosinophils from normal donors. Specific inhibitors of those kinases were used to study the involvement of particular signaling pathways in the activation of FcαR and FcγRII on eosinophils.

Percoll was obtained from Pharmacia (Uppsala, Sweden). FMLP and OVA were purchased from Sigma (St. Louis, MO). Human serum albumin (HSA) and purified human serum IgG (without traces of other Igs) were 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. Human rIL-5 was a gift from Dr D. Fattah (Glaxo/Wellcome, Stevenage, U.K.). IL-4 was a gift from Dr F. Kalthoff (Sandoz Forschungsinstitut, Vienna, Austria). Rabbit polyclonal ERK1 (C-16), ERK2 (C-14), and p38 MAPK (C-20) antisera were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antiphosphotyrosine polyclonal MAPK and p38 MAPK Abs were obtained from New England Biolabs (Berverly, MA). Pharmacological inhibitors wortmannin, LY294002, PD98059, and SB203580 were purchased form BioMol (Plymouth Meeting, PA).

Blood was obtained from healthy volunteers from the Red Cross Blood Bank (Utrecht, The Netherlands). Mixed granulocytes were isolated from the buffy-coat of 500 ml blood anticoagulated with 0.4% (w/v) trisodium citrate (pH 7.4) as previously described (28). 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 granulocytes were washed and resuspended in RPMI 1640 (Life Technologies, Paisley, U.K.) 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 PBS supplemented with 0.5% HSA and 13 mM trisodium citrate, and incubated with FMLP (10 nM) for 10 min at 37°C to decrease the specific gravity of the neutrophils, but not that of the eosinophils. Subsequently, eosinophils were obtained by centrifugation (20 min at 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 incubation buffer (20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgSO₄, 1.2 mM KH₂PO₄, supplemented with 5 mM glucose, 1.0 mM CaCl₂, and 0.5% (w/v) HSA). Purity of eosinophils was 97% (±0.5 SEM), and recovery was usually 80–90%. This procedure leads to the isolation of relatively unprimed eosinophils compared with conventionally used isolation procedures with immunomagnetic beads (29).

Eosinophils were isolated as described above and incubated at 37°C for 30 min. After pretreatment with or without PD98059, SB203580, LY294002, or wortmannin and stimulation with IL-4 or IL-5, cells (2 × 10⁶ cells) were washed twice in ice-cold PBS and lyzed in lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 40 mM β-glycerophosphate, 1% Triton X-100) supplemented with Na₃VO₄ (1 mM), aprotinin (10 μg/ml), leupeptin (10 μg/ml), and 1 mM PMSF. Lysates were precleared for 30 min at 4°C with protein A-Sepharose beads and subsequently, MAPK was immunoprecipitated with 1 μg of ERK1, ERK2, or p38 polyclonal antisera for 1 h at 4°C on a rotating wheel. Protein-A Sepharose was then added for a further 1 h at 4°C. After washing twice with lysis buffer with phosphatase inhibitors, samples were washed twice with kinase buffer (30 mM Tris-HCl, pH 8.0, 20 mM MgCl₂, 2 mM MnCl₂, 10 μM rATP, 10 μM MBP) without ATP. Precipitates were then incubated in 25 μl kinase buffer with 0.3 μCi [γ-³²P]ATP for 20 min at 30°C. Reaction was stopped by the addition of 5× Laemmli sample buffer. Samples were separated by electrophoresis on 15% SDS-polyacrylamide gels. MBP phosphorylation was detected by autoradiography.

Eosinophils were isolated as described above and incubated at 37°C for 30 min in incubation buffer. For detection of phosphorylation of p38 MAPK, eosinophils (0.5 × 10⁶ per condition) were washed twice in ice-cold PBS after stimulation and lyzed in lysis buffer (1% Triton-X100, 50 mM Tris-Cl, pH 8.0, 100 mM NaCl) with phosphatase inhibitors and subsequently boiled for 5 min after addition of 5× sample buffer. Total cell lysates were analyzed on 15% SDS-polyacrylamide gels. Proteins were transferred to Immobilon-P and incubated with blocking buffer (Tris buffered saline/Tween 20 (TBST) supplemented with 1 mM EDTA and 0.6% BSA) with polyclonal phospho-p38 MAPK antisera. Detection was with ECL (Amersham, Rainham, U.K.). Blots were reprobed with p38 MAPK Ab.

Serum IgG and serum IgA were coated to uncoated magnetic dynabeads (M-450, Dynal, Norway). Beads were washed twice with PBS (pH 8.5) and brought to a concentration of 45 mg/ml. Proteins were added at a final concentration of 1 mg/ml to the beads and mixed overnight at 4°C. The next day the beads were washed with borate buffer (0.5 M NaCl, 0.2 M H₃BO₃, and 0.02 M NaOH, pH 8.6) and blocked with 0.1 M lysine monohydrochloride (pH 8.6) in borate buffer for 2 h at room temperature. After two washes with 0.1 M acetate buffer (pH 4), beads were washed once with PBS with 1% (w/v) BSA. Until use, the beads were stored at 4°C in PBS/BSA at a concentration of 30 mg/ml (4 × 10⁸ beads/ml). Before the rosette assay, beads were resuspended in 20% (w/v) HSA and left for 20 min at room temperature.

Purified eosinophils were washed with Ca²⁺-free incubation buffer containing 0.5 mM EGTA and brought to a concentration of 8 × 10⁶ cells/ml. A 50 μl cell suspension (0.4 × 10⁶ cells) was incubated at 37°C. For priming, IL-5 and IL-4 were added 1:10, with final concentrations of 10⁻⁹ M. Cells were incubated with IL-4 for 5 min and with IL-5 for 15 min at 37°C. After priming, the beads were added in a ratio of 3.5 beads/cell. Cells and beads were mixed briefly and pelleted for 15 s at 100 rpm. Normally, eosinophils were incubated with beads during 30 min at 37°C. In the case of IL-4 primed eosinophils, beads and cells were incubated together for only 20 min. Longer incubation resulted in lower levels of binding, due to the transient effect of IL-4 priming, as described previously (19).

After incubation cells were resuspended vigorously and rosettes were 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 were 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. Previously we have demonstrated that the rosetting method with magnetic beads is very specific since 1) there is no appreciable background binding of eosinophils to control beads coated with OVA and 2) relevant blocking mAbs against Fc receptors inhibit the response (19).

For priming-inhibition studies, cells were pre-incubated with specific inhibitors before priming with cytokines. Cells were incubated with PI3K inhibitors wortmannin and LY294002 for 15 min at final concentrations of 20 nM and 1 μM, respectively. The p38 inhibitor SB203580 was incubated for 15 min at a concentration of 1 μM, while incubation with the MEK inhibitor PD98059 was for 30 min at a concentration of 10 or 50 μM.

Results were expressed as means ± SE. Statistical analysis was performed by using paired and unpaired Student’s t tests. p values < 0.05 were considered as statistically significant.

IgA and IgG receptors on human eosinophils have been shown to be modulated differentially by priming with Th2-derived cytokines IL-4 and IL-5 (19). Stimulation of human eosinophils with optimal concentrations of both IL-5 (10⁻⁹) and IL-4 (10⁻⁸) leads to a time-dependent, transient activation of the lipid kinase PI3K, as measured in antiphosphotyrosine immunoprecipitates (30). Preincubation with 30 nM wortmannin, a relatively specific PI3K inhibitor, resulted in a complete inhibition of PI3K activation in eosinophils stimulated with IL-5.

To investigate the involvement of PI3K in the cytokine-induced Fc receptor activation on eosinophils, we performed rosette assays in the presence of wortmannin or LY294002, which both inhibit PI3K activity through distinct mechanisms. Freshly isolated eosinophils from normal donors only bind very weakly human serum IgA-coated beads. Only 8% of the cells are able to form rosettes (19). However, as shown previously, priming of eosinophils with IL-4 or IL-5 dramatically changes the ability of these cells (50–60%) to bind IgA-coated beads (Fig. 1,A, open bars) (19). The binding of IgG-coated beads to unprimed cells (25%) is also increased by pre-incubation with IL-5, but IL-4 does not significantly change the capacity of eosinophils to bind IgG-beads (19). As shown in Fig. 1,A, blocking of PI3K activation with wortmannin (gray bars) or LY294002 (filled bars) completely inhibited the rosette formation of IgA-coated beads with IL-4 or IL-5 primed eosinophils. We have previously demonstrated that the concentrations of wortmannin and LY294002 used here inhibit PI3K in human eosinophils (30). This suggests a role for PI3K in the cytokine-induced priming of IgA receptor functioning. In contrast, the ability of IL-5 primed eosinophils to bind IgG beads is not influenced by incubation with wortmannin or LY294002. (Fig. 1 B). These results show that activation of the PI3K pathway is involved in the specific priming of IgA-binding, but not crucial for IL-5 induced priming of IgG-binding.

We have also studied the activation of ERK MAPKs to determine their role in Fc receptor stimulation. First, we analyzed the ability of IL-4 and IL-5 to induce activation of MAPKs ERK1 and ERK2, which are both downstream targets of p21^ras signal transduction.

To measure the kinase activity of both ERK1 and ERK2 after stimulation, we specifically immunoprecipitated ERK1 or ERK2 and performed a kinase assay as described in Materials and Methods. As shown in Fig. 2, ERK1 kinase activity was minimally effected by stimulation with IL-4 or IL-5, which is in agreement with the unaltered levels of phosphorylation of ERK1 in eosinophils (30). Kinase activity of ERK2 was clearly induced by IL-5 (10⁻⁹ M) stimulation but remained unaffected by IL-4 (5 × 10⁻⁹ M) (Fig. 2). We have utilized a pharmacological inhibitor of the p21^ras-ERK pathway, PD98059, which has been described as a specific inhibitor of MEK, the upstream kinase mediating activation of ERK1 and ERK2 by p21^ras (31, 32). Pretreatment of eosinophils with PD98059 (50 μM) resulted in an inhibition of ERK2 activation after IL-5 stimulation as measured by MBP phosphorylation (Fig. 2, lower panel, lane 6).

It is clear that ERK2 is activated in eosinophils after stimulation with IL-5 and this activation can be inhibited by PD98059. We thus studied the effect of the MEK inhibitor on Ig-rosette formation with eosinophils to investigate whether the activation of ERKs is involved in the modulation of Ig receptors.

Interestingly, whereas PI3K appeared to be critical for binding of IgA beads (Fig. 1,A), activation of ERKs is not required for FcαR activation. Binding of IgA-coated beads to primed eosinophils was not inhibited by preincubation with 10 μM of the MEK inhibitor, PD98059 (Fig. 3,A, gray bars). Even a higher concentration, 50 μM, which completely inhibits ERK activation (Fig. 2) does not significantly effect rosette formation with IgA beads (Fig. 3,A, filled bars). However, 30-min incubation with PD98059 before IL-5 stimulation resulted in a 60% decrease of IgG binding. Levels of IgG binding to PD98059 treated and IL-5-stimulated cells were reduced to the levels of IgG binding to unprimed eosinophils (Fig. 3 B).

Although p38 MAPK was initially described as being activated by a variety of cellular stresses, it is now also known to be activated in response to various inflammatory cytokines (33, 34, 35). We thus studied the stimulation of p38 MAPK phosphorylation in human eosinophils. Phosphorylation of p38 after stimulation with IL-4 and IL-5 was detected by Western blot analysis with an activation-specific Ab that only interacts with Tyr¹⁸²-phosphorylated p38. As shown in Fig. 4,A, upper panel, a phosphorylated form of p38 is clearly detected in eosinophils that are stimulated for 15 min with IL-5. However, stimulation with IL-4 induced a rapid but very weak, transient phosphorylation of p38 MAPK. In all donors tested, the phosphorylation of p38 had already diminished after 5-min stimulation with IL-4. This in contrast with p38 phosphorylation by IL-5 that remained for at least 25 min (data not shown). Reprobing of the blot with p38 antiserum (Fig. 4,A, lower panel) shows an equal amount of p38 protein in all lanes. Activation of p38 MAPK is also shown in Fig. 4,B. Although the level of p38 MAPK activity in unprimed cells was variable (compare first lanes of both panels in Fig. 4,B and first lane in Fig. 6), an increase of p38 MAPK activity was detected both in IL-4- and IL-5-stimulated cells. Cytokine induced p38 MAPK activation could be blocked completely by 1 μM of SB203580 (Fig. 4 B, right lane of both panels). This inhibitor, a pyridinyl imidazole compound, is a highly selective inhibitor of p38 activity and has been used extensively as a tool to evaluate p38 MAPK-dependent events in vivo (36).

To study a potential role for the activation of p38 kinase in activation of Fc receptors by Th2-derived cytokines, we studied the binding capacity of primed eosinophils after addition of SB203580. As shown in Fig. 5, inhibition of p38 in eosinophils results in a striking difference in binding capacities of the cells to either IgA or IgG beads. The effect of priming with both IL-4 and IL-5 on IgA binding appears to be abolished by incubation with SB203580. The effect of IL-4 priming on IgA rosette formation (Fig. 5,A) is dramatically decreased by pre-incubation with a 100 nM concentration of SB203580 (Fig. 5,A, middle panel, gray bar), and a higher concentration (1 μM) of the inhibitor totally blocks the effect of IL-4 on rosette formation (Fig. 5,A, middle panel, filled bar). IL-5-primed binding of IgA beads is already completely inhibited by the lower concentration (100 nM) of SB203580 (Fig. 5,A, right panel, gray bar). In contrast, IL-5-induced modulation of FcγRII on eosinophils is not affected by pre-treatment with SB203580 (Fig. 5 B).

As suggested by Figs. 1 and 5, both activation of PI3K and p38 MAPK are involved in the modulation of FcαR on human eosinophils. Since inhibition of either PI3K or p38 MAPK activity resulted in a complete loss of IgA rosette formation, we questioned whether PI3K and p38 MAPK may be components of a single signal transduction pathway activated upon Th2-cytokine stimulation. We investigated this possibility by performing p38 MAPK assays with IL-5-primed eosinophils that were pretreated with the PI3K inhibitors LY294002 and wortmannin. As shown in Fig. 6, activation of p38 MAPK by IL-5 is blocked by inhibiting PI3K activation by both LY294002 (1 μM) and wortmannin (30 nM), suggesting that indeed PI3K and p38 MAPK are activated in the same signal transduction pathway and that p38 MAPK is activated downstream of PI3K.

The phenotype of activated eosinophils in vivo is well described and much is known concerning the importance of the eosinophil in inflammatory diseases such as allergic asthma. It is also clear that cytokines and chemoattractants are important modulators of eosinophil functions in vivo, and several signal transduction pathways are activated in eosinophils after stimulation with these agonists (30, 37). However, little is known about the molecular mechanisms that finally lead to activation of eosinophils at specific sites of inflammation. The hypothesis that there might be spatial and temporal regulation of eosinophil priming by different cytokines is an interesting concept to understand eosinophil activation in patients with allergic diseases. As shown previously, receptors for IgA and IgG on human eosinophils are differentially modulated by Th2-derived cytokines IL-4 and IL-5 (19). In this study we show that IL-4 and IL-5 activate Ig receptors by means of different signal transduction pathways.

Binding of IL-5 to its receptor on eosinophils directly results in association and activation of members of the JAK/STAT pathway and phosphorylation and activation of several kinases (37, 38), including the tyrosine kinase Lyn (37) as well as PI3K (30). The IL-4R signals through a complex network of signaling molecules (reviewed in Refs. 39 and 40) including members of JAK/STAT pathway (41); PI3K (42); specific cellular substrates such as insulin receptor substrate-2 (43, 44) and Cbl (45); and protein kinases including Fes (46). In contrast with IL-5, IL-4 is unable to activate the p21^ras pathway, and ERK1 and ERK2 are not activated by IL-4 stimulation (47). Although tyrosine phosphorylation of multiple proteins and PI3K activation by IL-4 stimulation are described in several cells and cell lines, very little is known about IL-4 signaling in human eosinophils.

As previously described IL-4 and IL-5 are potent activators of PI3K (IL-4 and IL-5) and ERK2 (IL-5) (30), but this is the first report demonstrating p38 MAPK activation in eosinophils. p38 MAPK (also known as CSBP, RK, and HOG1) is a recently identified protein kinase that shares sequence similarity with other MAPKs. p38 MAPK is activated by the dual specific kinase MKK3 following exposure to products of microbial pathogens, environmental stress and pro-inflammatory cytokines (34, 48, 49). It has previously been shown to be activated by cytokines such as TNF-α, IL-1, and granulocyte-macrophage CSF in human neutrophils (36, 50, 51, 52).

This is the first report to show that IL-4 and IL-5 are able to activate p38 MAPK. Although p38 phosphorylation by IL-5 priming seems to be much stronger, both IL-4 and IL-5 are able to induce p38 kinase activity, as shown in Fig. 4. This demonstrates that downstream of the IL-4 and IL-5 receptors some signals converge at the level of p38 MAPK. The specific nature of these signals remains to be elucidated. To examine if there was a role of p38 in IL-4- and IL-5-induced activation of Fc receptors, we utilized the potent and highly selective inhibitor of p38 kinase, SB203580 (36). This inhibitor has been useful in dissecting signaling pathways involved in inflammatory responses. Our data suggest that p38 activation also plays a role in the activation of Ig receptors on human eosinophils. Cytokine-induced binding of IgA-coated beads to human eosinophils was blocked by inhibition of p38 activation (Fig. 5), since incubation of the cells with SB203580 before IL-4 or IL-5 stimulation leads to a complete inhibition in IgA binding. The kinetics of IL-4 priming on rosette formation with IgA beads correlate with the fast induction of p38 MAPK phosphorylation by IL-4. This is different for IL-5-induced p38 and ERK phosphorylation and IgA-binding, which is much slower and remains activated for a longer duration.

The fact that inhibition of either the p38 MAPK or PI3K pathway results in loss of IgA binding after priming demonstrates that both pathways are involved in modulation of the IgA receptor on eosinophils. It is tempting to speculate that p38 and PI3K might be both components of one signal transduction pathway that is activated by both IL-4 and IL-5 and utilized for the priming of FcαR activation. Since PI3K is a lipid kinase that is activated by translocation to the membrane, it is not likely that it will act downstream of p38. Activation of p38 by PI3K has previously been suggested by Krump et al. (53), and here we show that p38 MAPK activation can indeed be a downstream target of PI3K in eosinophils. Activation of p38 MAPK is dramatically decreased in eosinophils in which PI3K activity is blocked by either LY294002 or wortmannin (Fig. 6), suggesting that under these circumstances PI3K activation is necessary for proper p38 activation.

We have also shown that In contrast with IgA binding, inhibition of PI3K and p38 MAPK activity do not influence the effect of IL-5 on binding of IgG beads, suggesting that neither one of these pathways plays a major role in FcγRII modulation by IL-5. Modulation of FcγRII functioning seem to require other signals including the activation of the p21^ras-ERK pathway, since inhibition of this pathway with PD98059 results in a complete reduction of IgG rosette formation after stimulation with IL-5. Therefore, it is not surprising that IL-4 is not able to induce IgG binding to eosinophils (19), since IL-4 it cannot activate the p21^ras pathway (54).

In Fig. 7, a model is presented for the hypothesis that selective production of Th2-derived cytokines can lead to activation of different Ig receptors. This model predicts the distinction between FcαR and FcγRII activation and the general signal transduction pathways initiated by IL-5 compared with the more restricted activities of IL-4. The control of receptor activation by priming might be a critical process for the final activation of eosinophils. The regulation of Fc receptor affinity on eosinophils is analogous to the activation of integrins by diverse cellular stimuli. Both receptor types are controlled by inside-out signaling generated from cytokine/chemotaxis receptors. Although inside-out signaling is now well established for integrins (55), this is a novel concept for Fc receptors.

Despite the clear phenotype of cytokine-activated eosinophils in the context of Ig binding, not much is known about the mechanisms responsible. Several possibilities can be suggested: 1) a conformational change of the receptor, leading to an affinity switch to a higher affinity for Ig (56), and 2) membrane expression of receptors that are stored in secretory granules and released upon triggering of the cells with cytokines. It has been described for neutrophils that priming results in rapid mobilization of subcellular granules to cell surface to increase the number of granule associated receptors (57). 3) During activation of leukocytes, co-capping or clustering of integrin and Fc receptors can lead to an avidity switch (58). 4) Cytokine-induced activation of Ig receptors on eosinophils might be caused by inhibition of an inhibitory signal which keeps the receptor in a nonfunctional state. A suppressive regulation has been suggested to explain the default highly activated state of integrin α_vβ₃ when it is purified, in contrast with the moderate affinity state of cellular α_vβ₃ (59). The hypothesis of an “inside-out suppressor” is supported by the finding that adding protein kinase C inhibitors to eosinophils leads to increased responsiveness of opsonized particles (60). Additional experiments will be necessary to determine the precise nature of the signals mediating these effects.

In conclusion, activation of Fc receptors appear to be a tightly regulated process of inside-out signaling from cytokine receptors leading to receptor activation. Production of specific cytokines by Th2 lymphocytes leads to either a generalized priming phenotype (IL-5/PI3K, p38 MAPK, and ERK2) associated with activation of Ig receptors and integrin receptors or a restricted phenotype (IL-4/PI3K and p38 MAPK) leading primarily to enhanced IgA receptor functions. Elucidation of the mechanisms generated by the aforementioned receptor-activated signals will lead to insight into the mechanisms of eosinophil priming occurring in allergic diseases in vivo.