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(Journal of Leukocyte Biology. 2002;72:391-400.)
© 2002 by Society for Leukocyte Biology

Regulation of mediator secretion in human basophils by p38 mitogen-activated protein kinase: phosphorylation is sensitive to the effects of phosphatidylinositol 3-kinase inhibitors and calcium mobilization

Bernhard F. Gibbs, Katharina E. S. Plath, Helmut H. Wolff and Jürgen Grabbe

Department of Dermatology, Medical University of Lübeck, Germany

Correspondence: Dr. Bernhard F. Gibbs, Department of Dermatology, Medical University of Lübeck, Ratzeburger Allee 160, D-23538 Lübeck, Germany. E-mail: bfgibbs{at}gmx.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although human basophils modulate allergic diseases by secreting histamine, leukotriene C4, interleukin (IL)-4, and IL-13, the intermediary signals controlling the release of these mediators are poorly understood. Here, we show that p38 mitogen-activated protein kinase (MAPK) crucially affects basophil activation following stimulation with various secretagogues. Phosphorylation of p38 MAPK occurred within 5 min following anti-immunoglobulin (Ig)E stimulation, but was more rapidly activated in basophils stimulated with formyl-Met-Leu-Phe or A23187. Additionally, activation of p38 MAPK to the above stimuli was dependent on extracellular influx and intracellular mobilization of calcium. SB 203580, a specific p38 MAPK inhibitor, blocked anti-IgE-induced secretion of all basophil mediators and reduced not only p38 MAPK, but also extracellular signal-regulated kinases 1 and 2 activity, whereas the MAPK antagonist, PD 098059, did not affect p38 MAPK. IgE-dependent activation of p38 MAPK and MKK3/6 was affected by LY 294002 and wortmannin, suggesting that these kinases are targets for phosphatidylinositol 3 kinase (PI 3-K). We conclude that p38 MAPK is a pivotal regulator of basophil function downstream of PI 3-K activation and calcium mobilization.

Key Words: signal transduction • inflammatory mediators • IgE


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Human basophils potentially play an important role in allergic disease [1 , 2 ]. They have been shown to rapidly invade tissues such as the skin [3 ], lung [4 , 5 ], and nose [6 ] following acute allergic reactions in atopic dermatitis, allergic asthma, and rhinitis, respectively. Basophils secrete large amounts of histamine and leukotriene C4 (LTC4) upon immunoglobulin (Ig)E-receptor cross-linking, thus contributing to the classical symptoms of hypersensitivity reactions, and may also govern T-helper cell type 2 immunological responses by the production of interleukin (IL)-4 and IL-13, which are released within several hours after basophil activation [7 8 9 10 11 ]. They also participate in chronic allergic inflammation due to increased vascular cell adhesion molecule 1 expression [12 , 13 ] and subsequent promotion of further granulocyte infiltration into affected tissues.

The intracellular signaling events, which control the production and release of the above basophil-mediator groups, are, however, poorly understood. So far, it is known that IgE-receptor signaling involves first the activation of Lyn and Syk tyrosine kinases [14 ] followed by activation of phospholipase C (PLC) [15 16 17 ] and phosphatidylinositol 3 kinase (PI 3-K) [18 19 20 ], both of which appear to be essential for subsequent mediator release. We recently showed that PI 3-K crucially controls the IgE-dependent release of histamine and LTC4 as well as cytokine synthesis in human basophils [19 ]. In contrast to PI 3-K, however, we and other groups have demonstrated that extracellular signal regulated kinases 1 and 2 (ERK1&2), which are regulated downstream of PI 3-K, primarily govern LTC4 secretion but have little effect on histamine release or cytokine production [19 , 21 ].

One possible element of PI 3-K-regulated pathways, which has been shown as being phosphorylated upon IgE-receptor cross-linking in basophils, is p38 mitogen-activated protein kinase (MAPK) [21 ]. In mast cells, p38 MAPK is activated by PI 3-K [22 ] and is known to affect actin filament reorganization [23 ], which may be involved in degranulatory processes such as histamine release from basophils. This kinase has also been described as regulating arachidonic acid release in platelets by activating phospholipase A2 [24 , 25 ] and is seen as modulating cytokine synthesis by its effects on activating transcription factor (ATF)-2 transcription factor [26 27 28 ]. Based on this evidence in other cell systems, p38 MAPK may therefore govern the release of all major basophil mediator classes. Miura et al. [21 ] showed that basophil p38 MAPK is indeed activated following stimulation with anti-IgE or the bacterial peptide formyl-Met-Leu-Phe (fMLP) and that the kinetics are faster than that seen for ERK activation.

Our main aim in the current study was to characterize the role of p38 MAPK in basophil-mediator secretion, in particular with respect to IgE-mediated events. Further, using specific inhibitors of p38 MAPK (SB 203580), PI 3-K (LY294002), and ERK [the MAPK kinase (MEK) antagonist PD 098059], we investigated whether p38 MAPK is a target for PI 3-K and whether it affects the relatively more slowly phosphorylated ERK. A further aim of this study was to determine the role of calcium in the activation of p38 MAPK. Several reports have shown that PI 3-K is independent of calcium signaling in high-affinity IgE receptor (Fc{varepsilon}RI receptor) -bearing cells [29 30 31 ]. However, the secretion of histamine, LTC4, and cytokines is known to be calcium dependent and controlled by PI 3-K. Intracellular calcium mobilization is caused by the action of PLC-generated inositol triphosphatase (IP3) on adenosine 5'-triphospahte (ATP) -sensitive channels on the endoplasmic reticulum (ER), and in RBL-2H3 mast cell lines, PLC has been shown to be regulated by PI 3-K [16 ]. It is thought that intracellular calcium release from these stores subsequently opens ICRAC calcium channels, leading to the influx of the ion from the extracellular milieu, which is essential for mediator secretion (reviewed in ref [32 ]). Here, we show for the first time that calcium mobilization is essential for the phosphorylation of p38 MAPK, suggesting that PI 3-K, which also crucially governs the activity of this protein, may be involved in PLC regulation and subsequent IP3-mediated intracellular calcium release.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and materials
The following were purchased: anti-mouse Ig horseradish peroxidase (HRP) and ECL+plus (Amersham, Buckinghamshire, UK); LY 294002 and PD 098059 (Alexis, Grünberg, Germany); IL-4 enzyme-linked immunosorbent assay (ELISA; BioSource, Camarillo, CA); SB 203580, SB 202190, and wortmannin (Calbiochem, Bad Soden, Germany); IL-13 ELISA (Boehringer Ingelheim Bioproducts, Heidelberg, Germany); RPMI-1640 (Gibco-BRL, Uxbridge, UK); LTC4 ELISA (Immunotech, Hamburg, Germany); magnetic cell sorter (MACS) basophil isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany); anti-rabbit Ig HRP, rabbit anti-ERK1&2, rabbit anti-p38 MAPK, rabbit antiphospho MKK3/MKK6 (Ser189/207), rabbit anti-MKK3, and rabbit antiphospho p38 MAPK (New England Biolabs GmbH, Frankfurt am Main, Germany); Ficoll-Paque (Pharmacia Biotec, Upsala, Sweden); and anti-human IgE, mouse antidiphosphorylated ERK1&2, calcium ionophore A23187, fMLP, 2x concentrated Laemmli sample buffer, and recombinant human (rh)IL-3 (Sigma Chemical Co., St. Louis, MO). All other standard reagents were purchased at the highest grade obtainable.

Basophil purification
Human basophils were enriched from buffy coats (leukocyte concentrate of 450 ml venous blood), which were obtained from healthy blood donors undergoing routine blood donation. The purification procedure involved a three-step process consisting of Ficoll density centrifugation followed by elutriation and negative selection using a commercially available kit (MACS basophil isolation kit, Miltenyi Biotec), as we have previously described [33 ]. Basophils were 91.0 ± 2.5% pure, as determined by alcian blue staining, with a mean yield of 3.2 ± 0.3 x 106 basophils per donor and more than 94% viable cells using trypan blue exclusion. These purities were sufficient to reliably measure mediator secretion from basophils with negligible contribution from other cell contaminants, as previously reported [7 , 8 , 34 , 35 ]. For immunoblotting experiments, near-homogeneous basophils (purity range, 96–100%) were used. In earlier studies, we have shown that there are no functional differences between purified and unpurified basophils using the above negative selection technique for basophil enrichment [36 ].

Kinase inhibitors
SB 203580, SB 202190, LY 294002, PD 098059, and wortmannin were dissolved in dimethyl sulfoxide (DMSO) at 0.1 M and subsequently diluted in RPMI-1640 buffer shortly before use. At working dilutions of the drugs (10 µM or less), no effects were observed as a result of DMSO in the absence of the drugs.

Cell stimulation and mediator assay
Purified basophils (1–2x105 nucleated cells per ml) were resuspended in RPMI-1640 medium (400 µl per tube), and following a short warming period (5 min), cells were preincubated for 15 min with various concentrations of the kinase antagonists before stimulation with anti-IgE (1/50000 dilution unless stated otherwise), rhIL-3 (10 ng/ml), A23187 (5 µM), fMLP (100 nM), or thapsigargin (1 µM). We used a suboptimal concentration of anti-IgE (1/50000 dilution), since we have seen that this gave rise to significant release of all basophil mediators on the linear part of the titration curve for each mediator. This is important to note, as IL-4 and IL-13 secretions from basophils are suppressed by higher anti-IgE concentrations, which cause maximum histamine release [7 ]. Similar bell-shaped, dose-dependent effects are seen using anti-IgE antibodies from other sources, and it is recommended to perform a full titration curve in order to establish a working, suboptimal concentration. In some experiments, basophils were preincubated with IL-3 for 15 min before further stimulation with anti-IgE. Controls consisted of cells incubated with the stimulus in the absence of the inhibitors as well as cells incubated in buffer alone. In mediator release experiments, cells were stimulated for 30 min for histamine and LTC4 release or for 5 h for the secretion of IL-4 and IL-13. Cells stimulated for 30 min were centrifuged, and a portion of the supernatants (100 µL) was assayed for LTC4 content by ELISA (minimum sensitivity, 10.3 pg/ml), according to the manufacturer’s instructions. Histamine contents were measured spectrofluorometrically in the remaining supernatants together with the cell pellets, which were diluted accordingly and lysed with perchloric acid (4%). The percentage histamine release for each tube was determined from the total histamine content in the sum of pellet and supernatant tubes. The net histamine and LTC4 releases were calculated by subtracting the spontaneous from the stimulated releases. Cells incubated for 5 h were centrifuged, the respective release of IL-4 and IL-13 in the supernatants assayed by ELISA (according to the manufacturer’s instructions), and the results calculated as described earlier. The minimum sensitivity of the ELISA was 3 pg/ml for IL-4 and 1 pg/ml for IL-13. In some experiments, cytokine contents were assayed in the supernatants and lysed cell pellets (3x freeze-thaw cycles and debris removed by centrifugation). In these experiments, actinomycin D (2 µg/ml)-treated basophils were stimulated with anti-IgE to compare the relative amounts of cytokines in pellets and supernatants following abrogation of protein translation. Inhibition of mediator secretion was expressed as a mean percentage reduction of control (net mediator release induced by the stimulus without inhibitor) ± SEM. Statistical significances were assessed using a paired Student’s t-test.

Western blot analysis
Highly pure (>96% pure) basophils (2–3x105/sample) were treated as indicated and incubated at 37°C for varying times. In some experiments, basophils were resuspended and incubated in calcium-free HEPES-Tyrode’s solution (which was, in this case, also used to dilute drugs and stimuli) in parallel to cells incubated with calcium-containing HEPES-Tyrode’s solution. Reactions were terminated by briefly placing tubes in ice, after which all tubes were immediately centrifuged, supernatants removed for histamine analysis (to gauge the response), and cell pellets lysed by vigorous mixing in 15 µl lysis buffer [50 mM Tris-HCl pH 7.5, 5 mM ethylenediaminetetraacetate, 10 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid, 5 mM dithiothreitol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml aprotonin, 20 µg/ml leupeptin, and 10 mM benzamidine]. Lysed cell pellets were then stored at -80°C prior to Western blotting. Samples were mixed with an equal volume of 2x concentrated Laemmli sample buffer and boiled for 3 min. Proteins were separated by 12% sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes. To determine the molecular weight (m.w.) of the resolved proteins, m.w. rainbow markers were used for each SDS-PAGE run. Membranes were blocked for 4 h in 5% skimmed milk in Tris-buffered saline/Tween 20 (TBST) buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, 0.1% Tween 20) with gentle agitation. Following 3 x 5-min washes in TBST, membranes were incubated overnight (at 4°C) with specific mouse or rabbit antibodies to diphosphorylated ERK1&2, ERK1&2, phospho p38 MAPK (Thr180- and Tyr182-phosphorylated residues), p38 MAPK, phospho MKK3/MKK6, or MKK3, after which membranes were successively washed (4x5 min; TBST) and incubated with anti-mouse Ig HRP or anti-rabbit Ig HRP for 2 h with gentle agitation. Membranes were then washed (4x5 min; TBST), and proteins were visualized by autoradiography according to the manufacturer’s instructions (ECL+plus, Amersham International). Comparisons of phosphorylated (activated) ERK and p38 MAPK were made not only on the basis of equal cell numbers but also by observing the even signal intensity of nonphosphorylated ERK, MKK3, and p38 MAPK. After detection, membranes were stripped for 10 min using Re-blot plus reagent (Chemicon International, El Segundo, CA), washed in TBST (4x5 min), and reprobed. Immunoblots, showing the effects of inhibitors in anti-IgE-stimulated basophils, were also assessed using densitometry. Results are expressed as a percentage of band intensity caused by anti-IgE stimulation alone.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Phosphorylation of p38 MAPK in human basophils following stimulation with anti-IgE and IL-3
In agreement with an earlier study by Miura et al. [21 ], anti-IgE stimulation of human basophils resulted in the rapid phosphorylation of p38 MAPK (Fig. 1A ), which was maximal after 5 min and thereafter, and only slowly subsided at 30 min. In this study, we also investigated the effect of IL-3, a well-characterized, potentiatory factor of IgE-dependent stimulation, which produced some phosphorylation of p38 MAPK over basal levels, but this was much weaker than for anti-IgE stimulation. The kinetics of IL-3-induced p38 MAPK activation was more variable compared with anti-IgE stimulation: in five donors, maximum p38 MAPK phosphorylation was seen after 5 min, whereas in an additional three donors, maximum expression was achieved only after 15 min stimulation. A short preincubation with IL-3 potentiated anti-IgE p38 MAPK activation and considerably enhanced histamine release (Fig. 1B) . In some experiments (as shown in Fig. 1A ), the duration of anti-IgE-induced p38 MAPK phosphorylation was also prolonged in the presence of IL-3 to more than 30 min, but this was not so pronounced with other donors. IL-3 by itself at 10 ng/ml did not cause marked basophil-histamine secretion alone, except for a slight, though significant, increase in the release of the amine after 30 min. In addition to these experiments, we observed that IgE-dependent p38 MAPK phosphorylation increased in a dose-dependent manner with increasing anti-IgE concentration (Fig. 2A ); this was also mirrored by the secretion of histamine (Fig. 2B) , and total p38 MAPK remained constant.



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  		Figure 1. Time-dependent activation of p38 MAPK (A) and histamine release (B) in purified human basophils stimulated with anti-IgE (1/50000 dilution), IL-3 (10 ng/ml), and in combination (15 min preincubation with IL-3, followed by anti-IgE stimulation for the indicated times). Cell pellets were lysed and subjected to Western blotting and supernatants (together with a lysed cell total) assayed for histamine as described in Materials and Methods. Total (nonphosphorylated) p38 MAPK blots are included as an indication of protein loading. Histamine data are expressed as mean percentage histamine release ± SEM for four separate experiments. *, **, and *** represent significant (P<0.05, P<0.01, P<0.001, respectively) differences between stimulated release and spontaneous secretion (control) using a paired Student’s t-test.

 


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  		Figure 2. Anti-IgE dose-dependent phosphorylation of p38 MAPK (A) and histamine release (B) in basophils. The total p38 MAPK shows equal protein loading. Histamine release is expressed as mean percentage histamine release ± SEM for four separate experiments. * and ** represent significant differences (P<0.05 P<0.01, respectively) between stimulated release and spontaneous secretion (0) using a paired Student’s t-test.

 
Effect of SB 203580 on histamine, LTC4, IL-4, and IL-13 secretion induced by anti-IgE
As seen in Figures 1 and 2 , basophil-histamine release closely parallels p38 MAPK activation. To clarify the importance of the kinase in basophil mediator release, we investigated the effects of the specific p38 MAPK inhibitor, SB 203580, which specifically blocks enzyme activity by binding to the ATP pocket of the kinase without direct action on other kinases of the MAP family [36 37 38 ]. We observed that SB 203580 was a potent and efficacious inhibitor of histamine and LTC4 release (Fig. 3A ), producing more than 50% inhibition at 1 µM. Additionally, the inhibitor produced similar antagonistic effects on IL-4 and IL-13 secretion (Fig. 3B) , thus indicating that p38 MAPK plays a major role in governing the production and release of all basophil mediator types.



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  		Figure 3. Inhibition of anti-IgE-induced histamine and LTC4 (A) as well as IL-4 and IL-13 (B) release from basophils as a result of the p38 MAPK inhibitor SB 203580. Cells were incubated for 15 min with the inhibitor before activation with anti-IgE for 30 min (histamine and LTC4) or 5 h (IL-4 and IL-13). Results are expressed as mean percentage inhibition of stimulated release in the absence of the drug ± SEM for six to eight separate experiments. Unblocked releases were 31.8 ± 4.4% for histamine, 4021 ± 595 pg/106 basophils for LTC4, 222 ± 57 pg/106 basophils for IL-4, and 44 ± 7 pg/106 basophils for IL-13. All data were corrected for spontaneous secretions (7±1% for histamine, 388±131 pg/106 basophils for LTC4, and less than 13 pg/106 basophils for IL-4/IL-13). *, **, and *** represent significant differences (P<0.05, P<0.01, P<0.001, respectively) compared with stimulated release in the absence of the inhibitor using a paired Student’s t-test.

 
As the inhibitory effect of SB 203580 was similar for histamine and cytokine secretion, it was possible that the drug merely blocks basophil degranulation rather than affecting the de novo cytokines synthesis. Therefore, we measured the levels of IL-4 and IL-13 after stimulation present in lysed cell pellets as well as supernatants and compared the effects of the drugs with that of the protein synthesis inhibitor actinomycin D (Fig. 4A and 4B ). Cytokine contents in cell pellets and supernatants in anti-IgE-stimulated basophils in the presence of actinomycin D were similar to that in unstimulated controls. However, omitting the inhibitor, anti-IgE stimulation alone not only increased cytokine release from the cells but also increased cellular levels compared with unstimulated or actinomycin D-treated cells, probably because of a lag between cytokine synthesis and release. In the presence of SB 203580, a reduction of cell-associated and secreted IL-4 (Fig. 4A) and IL-13 (Fig. 4B) was observed. Therefore, these results suggest that SB 203580 inhibits IL-4 and IL-13 production at or before the level of protein translation and signify that these mechanisms are affected in parallel to basophil degranulation and leukotriene synthesis.



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  		Figure 4. Total levels of IL-4 (A) and IL-13 (B) in purified basophils measured in supernatants (open bars) and lysed cell pellets (solid bars). Cells were preincubated with actinomycin D, SB 203580, or buffer for 15 min before stimulation with anti-IgE together with control (buffer alone). Results are shown as mean cytokine content from five separate experiments.

 
To exclude the possibility that these inhibitory properties of SB 203580 were merely a result of toxic effects, we performed trypan blue exclusion tests on basophils incubated for more than 4 h in the presence or absence of the inhibitor. However, at 1 µM, SB 203580 had no significant effect on basophil viability (95.6±1.2% viability with SB 203580 compared with 97.2±1.7% in controls; n=5). Moreover, higher concentrations (10 µM) of SB 203580 as well as SB 202190 (which had similar effects on mediator secretion) did not substantially reduce basophil viability (>90%) and did not give rise to increased histamine release from the cells (data not shown).

Relationship between activation of p38 MAPK and PI 3-K as well as ERK
To elucidate where p38 MAPK may be acting with respect to other kinases known to regulate basophil activation, we employed the use of well-documented, specific inhibitors of not only p38 MAPK but also PI 3-K and MEK. As earlier studies showed that PI 3-K blockade prevents the release of all basophil mediator types [19 ], we first looked at whether p38 MAPK may be a target of PI 3-K using the specific inhibitor for this enzyme, LY 294002 as well as wortmannin, which is highly selective against PI 3-K below 100 nM. The results, shown in Figure 5 , demonstrate that PI 3-K inhibitors are effective antagonists of anti-IgE-induced p38 MAPK phosphorylation (Fig. 5A , lower panels). Additionally, we observed that these inhibitors blocked MKK3/MKK6 phosphorylation, which is known to directly activate p38 MAPK [39 40 41 ] using antibodies directed against phospho Ser189 and Ser207 residues on MKK3 and MKK6, respectively (Fig. 5A , upper panels). In contrast, the p38 MAPK inhibitor SB 203580 did not reduce MKK3/MKK6 activity but inhibited p38 MAPK phosphorylation.



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  		Figure 5. (A) Effects of LY 294002 and wortmannin on anti-IgE-induced MKK3/MKK6 (upper panel) and p38 MAPK (lower panel) activity in human basophils. Cells were preincubated with the drugs for 15 min before stimulation with anti-IgE for 5 min. Nonphosphorylated MKK3 and p38 MAPK are shown as loading controls. The Western blot is representative of five separate experiments. (B) Densitometric analysis of pooled data showing mean % intensities (of anti-IgE control) ± SEM.

 
The widely used specific MEK inhibitor PD 098059 was ineffective at abrogating p38 MAPK phosphorylation but, as expected, almost completely blocked ERK phosphorylation (Fig. 6 ). Conversely, SB 203580 reduced ERK activation, as did LY 294002, suggesting that signals controlling ERK phosphorylation may be targets of not only PI 3-K, as has previously been shown [19 , 20 ], but also p38 MAPK. SB 202190 (100 nM), a second specific p38 MAPK antagonist [36 , 42 ], had similar actions and inhibited mediator secretion as for SB 203580 and did not affect MKK3/MKK6 activity at this low concentration (results not shown).



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  		Figure 6. (A) Effect of SB 203580, LY 294002, and PD 098059 on anti-IgE-induced p38 MAPK and ERK1&2 phosphorylation in human basophils. Cells were preincubated with the drugs for 15 min before stimulation with anti-IgE for 10 min (which was shown to be suitable for the measurement of both activated kinses in parallel). Western blot is representative of three to four separate experiments. (B) Densitometric analysis of pooled data showing mean % intensities (of anti-IgE control) ± SEM.

 
Role of p38 MAPK in IgE-independent basophil secretion
When compared with the inhibition of anti-IgE-induced histamine release from basophils, SB 203580 was equally potent and efficacious at blocking release caused by the calcium ionophore A23187 (Fig. 7A ). Conversely, SB 203580 was less efficacious at reducing histamine release caused by fMLP, although the drug displayed similar potency. Despite the lack of inhibitory efficacy of SB 203580 in fMLP-stimulated basophils, the peptide caused a dose-dependent phosphorylation of p38 MAPK (Fig. 7B) . Thus, although p38 MAPK is activated by fMLP, the kinase may not play a crucial role in controlling downstream signals that lead to degranulation in fMLP-stimulated basophils compared with IgE signaling. The kinetics of fMLP-induced kinase activation also differed considerably from anti-IgE and is discussed in greater detail later (see Discussion).



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  		Figure 7. (A) Comparison between the effects of SB 203580 on basophil histamine release caused by anti-IgE and by A23187 (5 µM) or fMLP (100 nM). Cells were preincubated for 15 min with SB 203580 before stimulation with the secretagogues and, together with controls, incubated for an additional 30 min. Results are expressed as mean percentage inhibition of stimulated release in the absence of the drug ± SEM for eight separate experiments. Unblocked releases were 32 ± 4% for anti-IgE, 33 ± 5% for A23187, and 19 ± 4% for fMLP. All data were corrected for spontaneous secretions (5±1 %). *, **, and *** represent significant differences (P<0.05, P<0.01, P<0.001, respectively) compared with stimulated release in the absence of the inhibitor using a paired Student’s t-test. (B) Dose-dependent p38 MAPK phosphorylation in basophils caused by fMLP. The total p38 MAPK shows equal protein loading.

 
Effect of cyclic AMP (cAMP)-modulating drugs on p38 MAPK and ERK activity
With respect to the similar effects of SB 203580 on anti-IgE- and ionophore-induced histamine release, it may be deduced that calcium-sensitive signals play a role in p38 MAPK activation. Earlier studies by Botana and MacGlashan [43 ] have shown that a rise in cAMP prevents the influx of calcium ions into basophils and blocks mediator secretion. Our own studies have previously shown that cAMP-modulating drugs inhibit histamine and cytokine release from basophils following IgE-receptor activation [44 ]. Figure 8 shows that the ß-2 agonist salmeterol as well as the phosphodiesterase inhibitor theophylline reduce p38 MAPK phosphorylation. These findings prompted us to look more closely at the role of intracellular and extracellular calcium influx with respect to p38 MAPK activation.



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  		Figure 8. (A) Effect of salmeterol and theophylline on anti-IgE-induced p38 MAPK phosphorylation in human basophils. Total p38 MAPK is shown as loading control. Cells were preincubated with the drugs for 15 min before stimulation with anti-IgE for 10 min. (B) Densitometric analysis of pooled data showing mean % intensities (of anti-IgE control) from two separate experiments.

 
Role of calcium in basophil p38 MAPK activation caused by various secretagogues
To address the question concerning the role of calcium in p38 MAPK phosphorylation more thoroughly, we compared the kinetics of kinase activation, using various stimuli, in the presence and absence of extracellular calcium. Anti-IgE, IL-3, or a combination of both stimuli gave rise to a more pronounced p38 MAPK phosphorylation in the presence of extracellular calcium than in the absence of the ion (Fig. 9A 9B 9C ). In the absence of extracellular calcium, maximum p38 MAPK activation was reached after the same period of stimulation as cells stimulated in the presence of calcium, but in some donors, the signal returned more rapidly toward basal levels.



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  		Figure 9. Kinetics of anti-IgE (1/50000 dilution; A), IL-3 (10 ng/ml; B), and IL-3 + anti-IgE (C) induced p38 MAPK activation in the presence or absence of intracellular calcium. Total p38 MAPK is shown as loading control. Western blots are representative of three to four separate experiments.

 
A similar result was obtained for fMLP-induced p38 MAPK phosphorylation, although maximum kinase activity was less diminished in the absence of calcium compared with IgE-dependent stimulation. Noticeably, however, was the extremely fast kinetics of p38 MAPK activation, which was maximal following 1–5 min stimulation (Fig. 10A ). Interestingly, fMLP-induced p38 MAPK phosphorylation was short-lived even in the presence of calcium, although this was reduced further by its absence. This indicates that the control of mediator release may be linked to the longevity of p38 MAPK phosphorylation. The other stimuli used also led to a noticeable but brief activation of the kinase in calcium-free conditions without corresponding histamine secretion (data not shown), which suggests that degranulation caused by fMLP may be independent of p38 MAPK. This may also provide a reason why SB 203580 failed to efficaciously block fMLP-induced histamine release shown earlier (Fig. 7A) .



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  		Figure 10. Kinetics of fMLP (100 nM; A), A23187 (5 µM; B), and thapsigargin (1 µM; C) induced p38 MAPK activation in the presence or absence of intracellular calcium. (D) Effects of various incubation periods under the same conditions without stimulation. Total p38 MAPK is shown as loading control. Western blots are representative of two to three separate experiments.

 
In a similar manner to these observations, calcium ionophore A23187 gave rise to a strong phosphorylation of p38 MAPK in the presence of calcium, which was slightly diminished using calcium-free buffer (Fig. 10B) . A23187 transports calcium along its density gradient, and thus, the brief activation of p38 MAPK in calcium-free buffer may be ascribed to the release of the ion from intracellular stores. Given the similarity of effects of A23187 and other stimuli, it appears that mobilization of intracellular calcium as well as calcium influx play a crucial role in p38 MAPK activation. To prove this, we looked at the effect of thapsigargin, which specifically blocks a calcium ATPase channel pumping the ion into intracellular stores in the ER, thus increasing its cytoplasmic concentration. Figure 10C shows that thapsigargin, and therefore intracellular calcium, indeed can activate p38 MAPK in the absence of extracellular calcium. However, the agent produced a strikingly greater signal intensity and a more prolonged p38 MAPK phosphorylation in the presence of calcium in the buffer. This suggests that liberation of intracellular calcium causes the influx of the ion from the extracellular milieu and, given the similarities to the other secretagogues, that p38 MAPK is crucially regulated by this event.

As p38 MAPK is usually ascribed to stress-induced events in other inflammatory cells, we finally assessed whether calcium-free conditions increased spontaneous p38 MAPK activation in basophils incubated for various times (Fig. 10D) . The results showed that basophils incubated in the absence of stimulation exhibited even less spontaneous p38 MAPK phosphorylation in calcium-free conditions than in the presence of the ion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results presented here show that p38 MAPK is activated in basophils by various secretagogues and that it plays a major role in the production and release of proallergic mediators, particularly in regard to IgE-receptor activation. Until now, it has only been shown that p38 MAPK is phosphorylated upon anti-IgE- or fMLP-induced basophil activation [21 ]. The present study not only confirms these findings but also shows that p38 MAPK is an integral part of the PI 3-K-sensitive signaling network and is sensitive to extracellular calcium influx.

The actions of p38 MAPK in cellular signaling, particularly in inflammation, are not yet fully understood. The kinase (also known as SAPK2, RK, CSBP2, and HOG1) has been characterized mainly in terms of its involvement in cellular stress, such as UV light and osmotic shock [45 ]. Although it is not known whether these particular stress conditions affect p38 MAPK activation in basophils, the present study did show that subjecting the cells to a calcium-free environment does not increase the activity of this kinase. Furthermore, in a separate study, we have also observed that basophils exposed to high concentrations of hydrogen peroxide (>1 mM) lead to a striking reduction in IgE-dependent p38 MAPK phosphorylation (unpublished observations). Thus, although further studies are necessary, we have yet to find evidence to support that cellular stress gives rise to enhanced p38 MAPK activity in human basophils. In other histamine-containing cells, p38 MAPK has been shown to partially regulate IgE-dependent IL-4 transcription in RBL-2H3 cells [22 ], but has little effect on tumor necrosis factor {alpha} expression in MC/9 murine mast cells [46 ]. These reports also showed that p38 MAPK and the related MAPK JNK are regulated by PI 3-K, which underlines our own observations regarding PI 3-K controlling p38 MAPK in the present study.

Our data provide some indication that not only the magnitude but also the duration of p38 MAPK activation may be important for mediator secretion. This is inferred by two pieces of evidence. First, in the absence of extracellular calcium, basophils fail to release mediators, although p38 MAPK is still activated by various stimuli but only weakly or for a far shorter period than in the presence of calcium. Second, fMLP induces p38 MAPK phosphorylation for only 5 min (compared with longer activation periods with A23187 or especially anti-IgE), and SB 203580 only poorly blocks histamine release. This suggests that p38 MAPK regulates basophil degranulation only when it is phosphorylated for longer periods. It also shows that signals other than p38 MAPK may be involved in degranulation caused by IgE-independent activators such as fMLP. Several studies have reported that fMLP-stimulated basophils do not produce cytokines, such as IL-4 [8 , 47 ], which may be due to the only transient activation of p38 MAPK by the bacterial peptide. This is contrasted by the prolonged activation of p38 MAPK following IgE-dependent stimulation, leading to significant cytokine release from basophils that is sensitive to the p38 MAPK blockade, which highlights the importance of the kinase in cytokine generation.

Other than histamine and cytokine production, p38 MAPK was also seen as being involved in the generation of the lipid mediator LTC4. In an earlier study, we have shown that this eicosanoid is crucially regulated in basophils by ERK [19 ], an observation that others have confirmed [20 ]. Unlike inhibition of PI 3-K or p38 MAPK, blocking ERK phosphorylation caused by its upstream activator, MEK, leads only to substantial reduction of LTC4 synthesis while having less effect on histamine or cytokine release. We previously showed that PI 3-K antagonists also block ERK activity [19 ], and Miura and MacGlashan [20 ] showed that PI 3-K feeds into the Ras/Raf/MEK-MAPK pathway by affecting Ras. However, p38 MAPK and its upstream regulators MKK3/MKK6 were found to be affected by PI 3-K antagonists and are therefore a downstream target of PI 3-K signaling. As the p38 MAPK antagonist, SB 203580, reduced IgE-dependent ERK phosphorylation, p38 MAPK may therefore play an intermediary role between PI 3-K and the regulation of signals ultimately affecting ERK phosphorylation.

Other evidence suggests, however, that the hypothesis of a possible signaling cascade involving PI 3-K, p38 MAPK, and subsequent ERK activation may be too general or apply only to IgE-mediated events. IL-3, which by itself does not cause considerable mediator release from basophils alone, is a poor activator of p38 MAPK but gives rise to stark ERK phosphorylation [19 ]. IL-3-mediated ERK activation is only partially affected by the p38 MAPK inhibitor SB 203580 (unpublished observations), but less efficaciously than basophil stimulation with anti-IgE. One explanation for this may reside in previous reports showing that IL-3-generated signals are less calcium dependent than for IgE-dependent events [48 49 50 ]. This implies the participation of other signals, independent of p38 MAPK (which is sensitive to calcium), involved in IL-3-induced ERK activation.

A striking feature of p38 MAPK activation in basophils is its apparent dependency on calcium mobilization. The kinase was sensitive to blockade by cAMP-modulating drugs (which affect extracellular calcium influx) and phosphorylated more strongly in the presence of extracellular calcium than in its absence. Similar results were also seen with the intracellular calcium liberator thapsigargin, and the implications of these observations are twofold. First, it confirms earlier studies showing that a rise in intracellular calcium is linked to a further rise as a result of the influx of the ion through the plasma membrane (reviewed in ref [32 ]). From our data, given the kinetics of p38 MAPK phosphorylation caused by thapsigargin comparing calcium-free and calcium-containing buffer, it appears that this intracellular calcium mobilization may be sufficient alone to cause calcium influx. Second, our data highlight the importance of the ion in being necessary for IgE-, fMLP-, A23187-, and perhaps to a lesser extent, IL-3-dependent activation of p38 MAPK.

Given the crucial effects of calcium in p38 MAPK activation, this poses the question of whether PI 3-K, which appears to be an upstream regulator of p38 MAPK, acts by modulating calcium mobilization or by an independent mechanism. In the RBL-2H3 mast cell line, PI 3-K has recently been shown as being closely associated with the src family of tyrosine kinases linked to the IgE receptor [51 ] and is thus involved at a very early stage following IgE-receptor activation. On the one hand, Knoll et al. observed that neither intracellular calcium nor cAMP levels were affected by wortmannin in basophils activated by anti-IgE or fMLP [52 ], suggesting that PI 3-K is not a calcium regulator. Conversely, however, several reports show that PI 3-K does activate PLC in mast cells [16 , 18 ], leading to a subsequent rise in IP3 and intracellular calcium release. The question remains to what extent this takes place in basophils and whether it is an essential and sufficient step for subsequent p38 MAPK activation, ultimately governing degranulation, eicosanoid synthesis, and cytokine production.

In summary, we have shown that p38 MAPK is involved in basophil mediator generation and release, is sensitive to the actions of PI 3-K, but is not inhibited by the MEK antagonist PD 098059. The activation of p38 MAPK is also crucially controlled by a rise in intracellular calcium and its influx into the cells. A key role for p38 MAPK is indicated here, particularly following the biologically and clinically relevant IgE-dependent mechanisms, and may further our understanding of basophil signaling and provide possible targets for pharmacological intervention.


   ACKNOWLEDGEMENTS
 
The authors express their thanks to Prof. Holger Kirchner (Institute of Immunology and Transfusion Medicine, Medical University of Lübeck) for supplying buffy coats and to Frau Karin Dube for excellent technical assistance.

Received November 22, 2001; revised March 9, 2002; accepted April 1, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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