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Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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2 µM), but not
histamine release. Stimulation with anti-IgE Ab resulted in the
phosphorylation of ERKs, which was kinetically similar to both
histamine and LTC4 release and decreased toward resting
levels by 30 min. Similar to FMLP, PD98059 inhibited
anti-IgE-mediated LTC4 release (IC50, 2
µM), with only a modest effect on histamine release and IL-4
production at higher concentrations. Taken together, these results
suggest that ERKs might selectively regulate the pathway leading to
LTC4 generation by phosphorylating cPLA2, but
not histamine release or IL-4 production, in human
basophils. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI (high affinity IgE
receptor) or the bacteria product FMLP elicits the release of preformed
mediator (histamine) and de novo synthesized lipid mediator
(LTC4)3
(1, 2). FcRI-mediated stimulation also results in the production of
IL-4; however, FMLP is a poor stimulus for IL-4 (3, 4). In allergic
diseases (bronchial asthma, allergic rhinitis, and atopic dermatitis),
basophils infiltrate and secrete histamine and
LTC4 at specific reaction sites in the skin,
nose, and lung (5, 6, 7). Histamine and LTC4 derived
from basophils are thought to be the main factors of the late phase
allergic response. IL-4 induces cross-switching of IgG to IgE in B
lymphocytes and increases the expression of VCAM-1 on endothelial
cells, which promotes the infiltration of additional eosinophils and
basophils into inflamed tissues, suggesting that basophils also
contribute to chronic allergic inflammation. The signal transduction events leading to these mediator secretions are not well understood in human basophils. Histamine is a preformed mediator in granules and is released upon stimulation of various stimuli. LTC4 is a de novo synthesized lipid mediator that is converted from arachidonic acid (AA) by 5-lipoxygenase and LTC4 synthase (8). The production of LTC4 might be controlled by the availability of free AA (9). The cytokine, IL-4, is a newly generated protein that may involve increased transcription. These three distinct classes of mediators may be differentially regulated and secreted from human basophils. We and others previously reported several similar and dissimilar characteristics for secretion of these mediators from human basophils (1, 2, 3, 10).
The p42/p44 mitogen-activated protein kinases (MAPKs), referred to as extracellular signal-regulated kinases (ERK-2 and -1), transduce signals elicited via several receptors that either have intrinsic tyrosine kinase activity or are associated with nonreceptor tyrosine kinases (11). ERK-1 (p44MAPK) and ERK-2 (p42MAPK) are approximately 90% homologous, suggesting functional redundancy (12). ERKs are activated by MAP kinase kinases (MEKs), which phosphorylate the TEY domain on tyrosine and threonine residues (13). The phosphorylation of tyrosine and threonine residues in ERKs is required for their full activation (12). Several transcription factors have been identified in eukaryotic cells as targets of ERKs (14). For example, the induction of c-Fos is thought to be mediated by ERKs via phosphorylation of the transcription factor TCF/Elk 1 (ternary complex factor) (15). ERKs also activate cytoplasmic enzymes such as cytosolic phospholipase A2 (cPLA2), which is important in the release of AA (16).
The role of ERKs in the signaling cascade of events leading to the
release of inflammatory mediators and cytokines from human basophils
has not been reported. However, there is evidence that these kinases
are involved in the signal transduction pathways occurring with
FcRI-mediated activation of rodent mast cells. In rat basophilic
leukemia cells (RBL-2H3 cells), the aggregation of FcRI initiates a
cascade of events that includes the activation of
src-related tyrosine kinases, Lyn and Syk (17, 18, 19), and the
tyrosine phosphorylation of various proteins (20, 21). The activation
of ERKs is apparent from the tyrosine phosphorylation and shift in
electrophoretic migration of ERK-2 (p42MAPK) and, to a
lesser extent, of ERK-1(p44MAPK) as well as from an
increase in ERK activity (22). There have been several attempts to
associate the role of ERKs in degranulation, the release of AA, and the
production of TNF-
following FcRI-mediated stimulation (22, 23, 24).
More recently, Zhang et al. provided evidence that ERK-2
(p42MAPK) activation contributed to the release of AA and
the production of TNF-, but not to the release of hexosaminidase (a
marker of degranulation) (24). The causal association of ERK activation
with the release of AA and TNF- was based on studies with the
selective MEK inhibitor, PD98059 (24). Thus, it is likely that ERKs
regulate the pathways leading to the release of AA (by activating
cPLA2) and the production of TNF-, but not
degranulation, in IgE-dependent stimulation in RBL-2H3 cells. Their
role in regulating LTC4 formation in RBL-2H3
cells has not been fully elucidated because of the inability of these
cells to generate LTC4. However, the opposite
results for TNF- production were obtained in MC/9 murine mast cells
(25). Inhibition of ERKs with PD98059 had little effect on
FcRI-stimulated TNF- production in these cells. In contrast, the
MEKK1-regulated JNK pathway activated by FcRI was shown to be
mediated by this cytokine production (25). These two conflicting
results (RBL-2H3 cell data vs MC/9 murine mast cell clone data) suggest
that the role of ERKs may be different in distinct cells (even with the
same stimulation; FcRI-mediated stimulation). Therefore, the studies
using normal human cells to examine the role of ERKs in cell functions
should be important.
We have recently showed that the activation of cPLA2 is associated with free AA generation and LTC4 release in human basophils (26). Although there is conflicting evidence for the enzymatic source of AA in IgE-mediated stimulation of human basophils (26, 27), our most recent evidence favors the view that activation of cPLA2 is more closely associated with the AA generated and used for LTC4 formation. However, the causal relationship between cPLA2 activation and AA/LTC4 generation has yet to be determined, since there is no selective inhibitor for cPLA2. ERKs are known to phosphorylate and activate cPLA2 and may regulate transcriptional factors for cytokine production, but these have not been examined in human basophils.
Recently, it has been shown that another MAPK family member, p38 MAPK (the mammalian homologue HOG1 from yeast), rather than ERKs may have a role in the regulation of release of AA by activating cPLA2 in a particular cell type, platelets (28, 29, 30, 31). Moreover, p38 MAPK is known to regulate a transcriptional factor (ATF-2), thereby mediating new protein synthesis, including cytokines (32, 33, 34).
The current studies have examined the characteristics of ERK1/2 and p38 MAPK activation in human basophils, and their relationship to the secretion of the three major classes of mediators (histamine, LTC4, and IL-4). We show that ERK activation is responsible for the release of LTC4 from human basophils, but appears to have little if any role in histamine and IL-4 secretion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following were purchased: PIPES, BSA, EGTA, EDTA, D-glucose, NaF, Na4P2O7, Na3VO4, 2-ME, Nonidet P-40, and Tris-HCl (Sigma, St. Louis, MO); crystallized human serum albumin (HSA; Miles Laboratories, Elkhart, IN); FCS and RPMI 1640 containing 25 mM HEPES and L-glutamine (BioWhittaker, Walkersville, MD); Percoll (Pharmacia, Piscataway, NJ); Tris and Tween-20 (Bio-Rad, Hercules, CA); leupeptin, DTT, and PMSF (Boehringer Mannheim, Indianapolis, IN); rabbit anti-ERK-1 Ab, rabbit anti-ERK-2 Ab, and rabbit anti-p38 MAPK Ab (Santa Cruz Biotechnology, Santa Cruz, CA); anti-phosphotyrosine mAb (4G10; Upstate Biotechnology, Lake Placid, NY); rabbit anti-phospho-ERK (MAPK) Ab, recombinant ERK-2 (p42MAPK) protein, rabbit anti-phospho-p38 MAPK Ab, and biotinylated m.w. markers (New England Biolabs, Beverly, MA); peroxidase-linked donkey anti-rabbit Ig Ab and peroxidase-linked sheep anti-mouse Ig Ab (Amersham, Arlington Heights, IL); and PD98059 (Calbiochem, La Jolla, CA). Rabbit anti-cPLA2 Ab was provided by Dr. Lisa Marshall (SmithKline Beecham, King of Prussia, PA). Goat anti-human IgE was prepared as previously described (1).
A stock solution of PD98059 was prepared in DMSO. Controls were incubated with an equal concentration of DMSO.
Buffers and media
PIPES-albumin-glucose (PAG) buffer consisted of 25 mM PIPES, 110 mM NaCl, 5 mM KCl, 0.1% glucose, and 0.003% HSA. PAGCM was PAG supplemented with 1 mM CaCl2 and 1 mM MgCl2. PAG-EDTA consisted of PAG supplemented with 4 mM EDTA. Countercurrent elutriation was conducted in PAG containing 0.25% BSA in place of 0.003% HSA.
Basophil purification
Basophils were purified from residual cells of normal donors undergoing leukapheresis using Percoll density gradient and countercurrent flow elutriation as previously described (3). In all experiments involving the use of Western blot analysis of phosphorylated proteins, basophil purities were >80% as determined by Alcian blue staining (35). Basophil preparations of >73% purity were also used in studies involving the use of MEK inhibitor, PD98059.
The expression of ERKs and p38 MAPK in human basophils was initially
performed on basophils further purified by positive selection as
previously described (26, 36). This entire procedure was carefully
performed at a temperature below 4°C and in the presence of PAG-EGTA
(50 µM) to minimize concerns that the positive selection technique
might induce changes in ERKs status. Cells were incubated for 10 min
with 1 µg/ml mouse anti-human IgE (TES-19, provided by Dr.
Frances Davis, Tanox) in the presence of 4 mg/ml normal human
IgG to block Fc
R. After a subsequent 20-min incubation with rat
anti-mouse IgG2a+b paramagnetic beads (8 µl/107
cells), the cells were passed through a MACS minicolumn (MACS system,
Miltenyi Biotec, Sunnyvale, CA). Flow-through cells were collected, and
the eluted basophil purities were between 98 and 99.4%.
Basophils are the only leukocytes in these preparations to release histamine and LTC4 in response to FMLP and anti-IgE (2). Studies also show that basophils are the major, if not the sole, source of IL-4 following IgE-dependent activation (3). As a result, basophil-enriched mixed leukocyte suspensions were used to evaluate the effect of PD98059 on histamine, LTC4, and IL-4 secretion. Venous blood from consenting donors was anticoagulated with 10 mM EDTA, and basophil-enriched suspensions were prepared by a double-Percoll density centrifugation, as previously described (37). The purity of the basophils in the cell suspensions ranged from 2–15%.
Western blot analysis of whole cell lysates
High speed cell pellets (14,000 x g, 5–10
s) were resuspended at 2 x 107 cells/ml in lysis
buffer (50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10 mM EGTA, 5 mM DTT, 1%
Nonidet P-40, 1 mM PMSF, 20 µg/ml leupeptin, 100 µg/ml aprotinin,
and 10 mM benzamidine). After 20 s of vortexing, the cell lysates
were kept on ice for 20 min and then microfuged for 15 min at 4°C.
Supernatant was collected as a protein extract containing lysed cell
components without nuclei. Extracts from an equal number of basophils
(2 x 105 cell equivalents/lane) were diluted with an
equal volume of 2x loading buffer (0.125 M Tris-HCl (pH 6.8), 4% SDS,
0.005% bromophenol blue, and 20% glycerol; NOVEX, San Diego, CA)
containing 0.05% 2-ME and subjected to 10% Tris-glycine gradient gel
electrophoresis (NOVEX). Gels were then transferred to pure
nitrocellulose membranes (Schleicher & Schuell, Keene, NH) with a Trans
Blot (NOVEX). Electrophoresis and transfers were performed according to
the manufacturer’s recommendations. After transfer, membranes were
immersed in TBST (50 mM Tris (pH 7.5), 0.15 M NaCl, and 0.05%
Tween-20) containing 5% nonfat dried skim milk (Carnation, Los
Angeles, CA) overnight to block nonspecific binding. Membranes were
then washed three times (5 min each time) with TBST. Immunoreactive
proteins were detected using anti-ERK-1 Ab, anti-ERK-2 Ab, or
anti-p38 MAPK Ab, which were diluted (at 0.5 µg/ml) in TBST
containing 1% skim milk. After a 4-h incubation, membranes were washed
with TBST and then incubated with peroxidase-conjugated anti-rabbit
Ig Ab for 1 h. After five 10-min washes, membrane-bound
anti-rabbit Ig Ab was visualized with ECL Western blotting
detection reagents (Amersham), and HyperECL luminescence detection film
(Amersham). The enhanced chemiluminescence film images were converted
to digital format with a URL digital camera, and the images
were analyzed with National Institutes of Health Image (Wayne Rasband,
National Institutes of Health, Bethesda, MD) (26, 36). Although the
comparisons were made on the basis of equal numbers of cells, the
protein contents of these samples were also found to be equal.
Phosphorylation of ERKs, p38 MAPK, and cPLA2
The phosphorylation of ERKs was assessed using three different techniques: 1) anti-phospho-ERK Ab (38), 2) anti-phosphotyrosine Ab (4G10) (39, 40), and 3) electrophoretic mobility shift, using anti-ERK-1 and anti-ERK-2 Abs (23, 39). The phosphorylation of p38 MAPK was assessed using anti-phospho-p38 MAPK Ab or anti-phosphotyrosine Ab (41, 42). The phosphorylation of cPLA2 was assessed using electrophoretic mobility shift with anti-cPLA2 Abs (16, 26). After stimulating basophils in PAGCM buffer, reactions were stopped by adding ice-cold PAG and were microfuged for 5–10 s. Cell pellets were immediately lysed in lysis buffer (20 mM Tris (pH 7.5), 2 mM EDTA, 2 mM EGTA, 5 mM DTT, 1 mM PMSF, 10 mM benzamidine, 100 µg/ml aprotinin, 200 µg/ml leupeptin, 50 mM NaF, 5 mM Na4P2O7, 1 mM Na3VO4, and 1% Nonidet P-40). Extracts containing equal basophil cell numbers (5 x 105 cell equivalents/lane) were diluted with an equal volume of 2x loading buffer and subjected to 10% Tris glycine gel (NOVEX). After electrophoresis (160 V and 1.5 h for detection of MAPKs phosphorylation, and 160 V and 3.5 h for cPLA2 phosphorylation), proteins were transferred to nitrocellulose membranes (as described above). The membranes were immersed overnight in TBST containing 2% BSA (for detection by 4G10) or in TBST containing 5% nonfat dry skim-milk (Carnation) for the others. Immunoreactive proteins were detected using 4G10 in TBST containing 1% BSA or anti-ERK-1 Ab, anti-ERK-2 Ab, anti-cPLA2 Ab, anti-phospho-ERK Ab, or anti-phospho-p38 MAPK Ab, which were diluted in TBST containing 1% skim milk for 4 h. After washing, the membranes were incubated for 1 h with horseradish peroxidase-conjugated anti-mouse Ab (for 4G10) or horseradish peroxidase-conjugated anti-rabbit Ab (for the other Abs). After washing, enhanced chemiluminescence detection was performed as described above. For kinetic studies, the same membranes were detected by anti-phospho-ERK Ab, anti-ERK-1 Ab, and anti-ERK-2 Ab or anti-phospho-p38 MAPK Ab and anti-p38 MAPK Ab. Between each detection, membranes were stripped with stripping buffer (62.5 mM Tris-HCl (pH 6.7), 100 mM 2-ME, and 2% SDS) for 1 h at 50°C. In preliminary experiments the techniques to assess phosphorylation of ERKs were simultaneously compared using phosphorylated and nonphosphorylated ERK-2 (p42MAPK) proteins. Both phospho-ERK Ab and 4G10 recognized only phosphorylated ERK-2. Although the anti-ERK-2 Ab recognized both phosphorylated and nonphosphorylated ERK-2, the phosphorylated protein migrated more slowly than the nonphosphorylated protein, indicating that this Ab might also be useful in assessing phosphorylation of ERK-2 by recognizing both the phosphorylated and the nonphosphorylated form.
LTC4 and histamine measurements
Fifty thousand basophils were challenged in a final volume of 100 µl of PAGCM at 37°C. The reactions were terminated with 900 µl of ice-cold PAG-EDTA, and the cells were then centrifuged in a microfuge at 14,000 rpm for 10 s. A RIA was performed using 100 µl of supernatant to determine LTC4 levels as previously described (1, 43). An additional 500 µl of supernatants was mixed with an equal volume of PAG to measure histamine by automated fluorometry (44). The percentage of total histamine release was calculated for the diluted supernatants after subtraction of spontaneous histamine release (45). Each condition tested was performed in duplicate.
IL-4 measurements
Fifty thousand basophils were challenged in a final volume of 100 µl of RPMI 1640 with 0.03% HSA (37°C, 5% CO2 incubator). After 4 h, the supernatants were harvested by centrifugation of microtiter plates (150 x g, 3 min). The supernatants were stored at -80°C until analysis. IL-4 measurements were performed using an in-house ELISA, as previously described (46). Each condition tested was performed in duplicate.
AA measurements
The mass of AA was determined via combined capillary gas chromatography-negative ion chemical ionization mass spectrometry using modifications of previously described procedures (47, 48). One hundred thousand cells were challenged in a final volume of 100 µl of PAGCM at 37°C. The reactions were terminated by the addition of 1 ml of acetone followed immediately by the addition of 15–20 ng of octodeuterated AA (as an internal standard). Reaction mixtures were transferred to glass vials and dried under dry N2. The carboxyl moieties of AA were converted to pentafluorobenzyl esters as previously described (47). Once derivatized, the residue was dissolved in 60 µl of dodecane for injection into the gas chromatograph. The measurement of released AA by gas chromatography-negative ion chemical ionization mass spectrometry was performed as previously described (47, 48). Each condition tested was performed in duplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether ERK-1, ERK-2, and p38 MAPK were present in
human basophils, basophils that were very close to 100% purity
obtained by anti-IgE positive selection were prepared as described
in Materials and Methods. This study included a comparative
analysis with the contaminating cells (mainly monocytes and
lymphocytes) that typically contaminate enriched basophil preparations.
The collected cells that washed through the magnetized mini-MACS column
yielded contaminating cells that contained 
1% basophils. The
subsequently collected basophils ranged in purity between 98–99.4%.
The cell lysates from the two populations were examined by Western
blotting (Fig. 1
). Nonphosphorylated ERK-2
(p42MAPK) protein was used as a positive control. The
anti-ERK-2 Ab recognized only ERK-2, while the anti-ERK-1 Ab
recognized ERK-1 and, to a lesser extent, ERK-2. The results indicated
that basophils and contaminating cells expressed essentially equivalent
levels of ERK-1/2 and p38 MAPK. The intensities of bands were assessed
by image analysis from three different experiments. Human basophils
expressed ERK-1 (130 ± 20% of contaminating cells), ERK-2
(97 ± 6% of contaminating cells; n = 3), and p38
MAPK (166 ± 12% of contaminating cells; n = 2).
Our previous studies indicate that cPLA2 is also expressed
at equivalent levels in basophils and contaminating cells (26). Thus,
for all the experiments examining the phosphorylation of ERKs, p38
MAPK, and cPLA2 by Western blot, cell suspensions of >80%
basophils were used to minimize the contribution from contaminating
cells.
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As noted above, basophils activated with FMLP release histamine
and generate free AA/LTC4, much like they do
following IgE-dependent activation. We have shown more recently that
FMLP also induces the phosphorylation of cPLA2 at a time
consistent with free AA/LTC4 generation (26). We
therefore examined the relationship between histamine release and free
AA/LTC4 generation with the time course of
FMLP-induced phosphorylation of ERKs. As shown in Fig. 2A, the phosphorylation of ERK-1 and -2 did
occur, as assessed by the three different Abs described in
Materials and Methods. In these experiments, phosphorylated
and nonphosphorylated ERK-2 proteins were used as a reference. Thus, an
electrophoretic mobility shift of ERK-1 and -2 was detected by
anti-ERK-1 and anti-ERK-2 Abs, respectively. An identical time
course in the phosphorylation of ERK-1 and -2 was detected by
anti-phospho-ERK Ab (Fig. 2A) and by
anti-phosphotyrosine Ab (4G10; data not shown). The phosphorylation
of ERK-1 and -2 following FMLP stimulation was modest at 30 s, but
a more significant level of phosphorylation was observed after 1 min,
with maximal phosphorylation seen after 5 min (Fig. 2, A and
B). By 15 min, phosphorylation had decreased toward basal
levels. The phosphorylation of cPLA2, the generation of
free AA, and LTC4 release coincided with or
followed the phosphorylation of ERK-1/2. Unlike ERK-1/2,
cPLA2 remained phosphorylated at 15 min. On the other hand,
histamine release was nearly complete (80 ± 1% of maximum
histamine release) within 30 s. These results are consistent with
our previous findings that there is no lag time for histamine
release, whereas there is a lag time (1 min) before the generation
of free AA/LTC4 following FMLP activation (47).
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In the next series of experiments, we examined the relationship
between the phosphorylation of ERKs and the release of histamine and
LTC4 in response to IgE-dependent stimulation. As
shown in Fig. 3, A and B, there
was no detectable phosphorylation of ERK-1/2 following anti-IgE
activation for 1 min. However, both ERKs were maximally phosphorylated
by 5 min, with levels decreasing toward baseline by 30 min. Unlike
activation with FMLP, histamine release followed the phosphorylation of
ERKs. The cPLA2 phosphorylation and
LTC4 release also followed or coincided with
ERK-1/2 phosphorylation. Anti-IgE-mediated phosphorylation of ERKs was
also transient compared with phosphorylation of cPLA2.
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were the results for a nonreleasing donor.
Anti-IgE-mediated phosphorylation of ERKs and cPLA2 was not
detected; there was also no LTC4 release (data
not shown). Taken together, anti-IgE-mediated ERK activation may be
associated with the pathway leading to histamine release and
LTC4 generation. Kinetics of phosphorylation of p38 MAPK following stimulation with FMLP or anti-IgE Ab
The kinetics of phosphorylation of p38 MAPK following stimulation
with FMLP or anti-IgE Ab were also examined. As shown in Fig. 4A, a significant phosphorylation of p38 MAPK
was observed following stimulation with FMLP. The kinetics were
slightly faster (a significant phosphorylation was observed at 30
s, with a maximum from 1–5 min and a decrease toward the basal level
by 15 min) than those of phosphorylation of ERKs. Similar results were
obtained with anti-phosphotyrosine Ab (4G10) blot (data not shown).
Anti-IgE Ab also induced modest phosphorylation of p38 MAPK at 1 min,
and maximum phosphorylation was seen from 5–15 min (Fig. 4B). By 30 min, the phosphorylation decreased toward basal
levels. Similar results were obtained with anti-phosphotyrosine Ab
blot (data not shown). Taken together with previous results, p38 MAPK
may contribute to mediator release in human basophils.
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As noted, MEKs (MAP kinase kinases) are dual specificity kinases that phosphorylate substrates (ERKs) on both Tyr and Thr residues and increase the catalytic activity of ERKs (51, 52). PD98059 is a well-known inhibitor of MEK (MAPK kinase). This compound blocks the activation (and phosphorylation) of MEK through an allosteric mechanism that does not involve inhibition of ATP binding (53). The potency and specificity of this inhibitor have been extensively documented in a variety of biologic systems (24, 53, 54, 55). It has been shown that IgE-mediated phosphorylation of ERK-2 and cPLA2 are specifically inhibited by PD98059 in RBL-2H3 cells (24).
As shown in Fig. 5A, this also appears to be
true in human basophils. The phosphorylation of ERKs induced by FMLP
was prevented by PD98059, whereas the phosphorylation of tyrosine
in other proteins, including 38 (p38 MAPK)-, 62-, 100-, and
115-kDa proteins was not significantly affected by this compound.
Similar to phosphorylated 38-kDa protein (p38 MAPK) in
anti-phosphotyrosine Ab blot, phospho-p38 MAPK Ab blot also
confirmed that PD98059 did not affect phosphorylation of p38 MAPK (Fig. 5B). The electrophoretic mobility shift of cPLA2
was also inhibited (Fig. 5A), suggesting that PD98059
specifically blocks the ERK-cPLA2 pathway but not the
pathway for p38 MAPK in human basophils.
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To evaluate the involvement of the MEK-ERK pathway in
LTC4 generation and histamine release, the effect
of PD98059 on the secretion of these mediators was tested. For these
experiments, basophil-enriched suspensions freshly prepared from blood
or cell suspensions derived from leukocyte packs were used (purity
ranged from 2–75%). PD98059 did not affect FMLP-induced histamine
release (Fig. 6A). In contrast, PD98059
inhibited FMLP-induced LTC4 release (76 ±
5% inhibition at 100 µM and 64 ± 16% inhibition at 10 µM),
whereas the same dilution of DMSO did not affect the release of
LTC4 (Fig. 6B). The IC50
for the inhibition of LTC4 by PD98059 was
approximately 2 µM, comparable to values (2–4 µM) reported for the
inhibition of MEK and ERK in other cell types (53, 54, 55). The effect of
PD98059 on FMLP-induced generation of free AA was also examined. In
this series of experiments, the cells were stimulated with FMLP for 5
min, since this amount of time results in maximal free AA generation
(Fig. 2B). FMLP-induced free AA generation was also
inhibited by PD98059 (57 ± 8% inhibition at 100 µM and 41
± 7% at 10 µM compared with DMSO control; n = 3).
Taken together, these results suggest that FMLP activation uses the
MEK-ERK pathway for free AA/LTC4 generation but
not histamine release.
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The kinetic studies, shown in Fig. 3, suggested that the
anti-IgE-mediated activation of ERKs may be associated with
histamine release as well as LTC4 release.
Further, ERKs also regulate several transcriptional factors (14) that
may potentially have a role in cytokine production from human
basophils. The effects of PD98059 on anti-IgE-mediated secretion of
histamine, LTC4, and IL-4 were examined (Fig. 7). Based on previous experiments to determine
the optimal concentrations of anti-IgE to induce histamine and
LTC4 release (3), or IL-4 production, 100 ng/ml
(for histamine and LTC4 release) and 10 ng/ml
(for IL-4 production) of anti-IgE were used. As observed for FMLP
activation, anti-IgE-induced LTC4 was
inhibited by PD98059 (85 ± 10% inhibition at 100 µM and
80 ± 14% inhibition at 10 µM), with an IC50 of
approximately 2 µM. In contrast, histamine release (11 ± 10%
inhibition at 100 µM) and IL-4 production (37 ± 7% inhibition
at 100 µM vs 14 ± 12% inhibition with DMSO control) were only
marginally inhibited by PD98059, suggesting that ERKs are not important
in histamine release or IL-4 production.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FMLP-induced histamine release preceded the phosphorylation of ERKs and was unaffected by inhibition of the MEK-ERK pathway by PD98059. These results strongly suggest that the activation of ERKs is not involved in FMLP-mediated degranulation. In contrast, the release of histamine and LTC4 induced by anti-IgE was consistent with the time course of phosphorylation of ERKs. The kinetics of histamine release induced by anti-IgE are slower than those of FMLP (1, 47). Previous data suggested that the sustained elevation in cytosolic calcium that follows IgE-mediated stimulation was associated with histamine release. However, histamine release only follows elevations in cytosolic calcium after an interval of 2–3 min, suggesting the need for signaling events that are slower to evolve. This rate-limiting event for histamine release remains unclear, but the slower activation of ERK-1/2 suggested one possibility. However, the inhibition of the MEK-ERK pathway by PD98059 only marginally affected histamine release, suggesting little causal relationship between histamine release and ERK activation. Taken together with the results for FMLP, ERKs appears to have little role in any receptor-mediated degranulation in human basophils. It is useful to note that similar observations have been made in RBL-2H3 cells. PD98059 inhibited the release of AA; however, degranulation (hexosaminidase release) was only slightly affected in RBL-2H3 cells (24).
The activation of ERKs also induces c-Fos expression (which constitutes
part of the AP-1 complex) (15), the inactivation of I
B (61), and
enhanced binding activity of cytokine transcriptional factors such as
NF-B and activating protein-1 (62). In RBL-2H3 cells, IgE-mediated
TNF- production is inhibited by PD98059, suggesting that the MEK-ERK
pathway is involved in the IgE-dependent production of this cytokine
(24). In contrast, the same compound does not affect the similar
IgE-dependent production of TNF- in distinct cells (MC/9 murine mast
cells), suggesting that the MEK-ERK pathway does not regulate this
cytokine. However, the other MAPKs, JNKs, have been shown to mediate
this cytokine (25). These conflicting data suggest that roles of ERKs
for production of cytokines appear to be dependent on cell type or
species. Therefore, it is important to determine the role of ERKs in
normal human cells. Human basophils produce IL-4, and there is no
evidence of TNF- production from human basophils. We observed only
minor inhibition of IL-4 production by PD98059 at 100 µM in human
basophils. Furthermore, the transient activation of ERK-1/2 is not
consistent with the time scale of IL-4 secretion. Since disaggregating
cell surface cross-links at times greater than 30 min stops IL-4
secretion, there appears to be a need for sustained signaling, and the
transient nature of ERK-1/2 phosphorylation is not consistent with this
requirement. Taken together, the activation of ERKs may not be involved
in IgE-mediated IL-4 production from human basophils. This belief is
further supported by the evidence that FMLP, which induces the
phosphorylation of ERKs, is a poor stimulus of IL-4 production from
human basophils (3). At this time, there is more evidence to suggest
that a transcription factor resembling the nuclear factor of activated
T cells (NF-AT) has a greater role in IL-4 generation by these cells
(63). It remains possible that the activation of ERKs may contribute to
the synthesis of other proteins.
We also demonstrated the expression and phosphorylation of p38 MAPK in human basophils. Kinetics studies revealed that phosphorylation of p38 MAPK is slightly faster than that of ERKs following stimulation of either anti-IgE or FMLP, suggesting that p38 MAPK may regulate or modulate mediator release in human basophils. The inhibition of ERK pathway by PD98059 abrogated AA/LTC4 generation without affecting phosphorylation of p38 MAPK. These results may suggest that ERKs rather than p38 MAPK are essential for the pathway generating AA/LTC4. The precise role of p38 MAPK in mediator release remains to be elucidated. We are currently investigating this issue.
In summary, the activation of ERKs appears to be a key step necessary for the phosphorylation and activation of cPLA2, which is important for liberating free AA for LTC4 generation in human basophils. However, ERKs have no apparent role in supporting histamine and IL-4 release.
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2 Address correspondence and reprint requests to Dr. Donald W. MacGlashan, Jr., Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224. E-mail address:
3 Abbreviations used in this paper: LTC4, leukotriene C4; AA, arachidonic acid; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; MEK, MAPK kinase; MEKK, MAPK kinase kinase; cPLA2, cytosolic phospholipase A2; HSA, human serum albumin; PAG, PIPES-albumin-glucose.
Received for publication July 13, 1998. Accepted for publication December 30, 1998.
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