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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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI (high affinity IgE
receptor) or agonists for G protein-coupled receptors (e.g., FMLP)
elicits the release of preformed mediators (histamine) and de novo
synthesized mediators (e.g., leukotriene C4
(LTC4)3
and IL-4) (1, 2), but the profile of mediators depends on
both the particular secretagogue and the cytokine environment. For
human basophils, a variety of cytokines (e.g., IL-3, IL-5, and GM-CSF)
mediates up-regulation of secretion, of which IL-3 is generally the
most efficacious (3, 4, 5). With the exception of
preparations of basophils from certain donors, these cytokines alone
are poor stimuli of basophil secretion (3, 4, 5). These
cytokines are also found at sites of allergic inflammation
(6, 7, 8, 9, 10), suggesting that they can modify allergic
reactions by priming basophil secretion as well as affecting the
function of other leukocytes. However, the mechanisms underlying the
priming effect of cytokines such as IL-3 in basophils are only
partially understood. For a variety of studies, we have used as a model of the priming effect induced by IL-3 the change in LTC4 release during stimulation with C5a. C5a is an excellent secretatgogue for histamine release, but for basophils from most donors it does not induce LTC4 release (11, 12, 13). A 5-min preincubation of the cells with IL-3 permits C5a to induce marked LTC4 release (12, 13). To understand how IL-3 brings about this change, we have examined some of the signaling steps involved in generating LTC4 in human basophils. Current evidence supports the view that cytosolic phospholipase A2 (cPLA2) is required for generation of the arachidonic acid (AA) used for LTC4 synthesis (14, 15). This enzyme is activated by the combined effects of its phosphorylation (for its enzymatic activity) and an elevation in cytosolic free calcium (for its binding to phospholipid pools) (16, 17). Thus, these two signals must be present at the same time for cPLA2 to hydrolyze phospholipids and produce AA. Previous studies have shown that C5a induces a relatively brief transient rise in the cytosolic calcium response ([Ca2+]i), lasting about 30–45 s (12). In addition, cPLA2 is phosphorylated, but measurable changes in its phosphorylation state are not observed for the first 30–45 s in human basophils (14). These data indicate that there is little overlap in the two signals needed for activation of cPLA2, and this lack of overlap most likely explains the inability of C5a to induce the generation of free AA and consequently LTC4 (14).
A short treatment of basophils with IL-3 leads to phosphorylation of cPLA2 (14). Previous studies led us to speculate that IL-3 may induce phosphorylation of ERK1/2, the antecedent kinases most likely responsible for the phosphorylation of cPLA2 (16, 18). We have also shown that a short treatment of basophils with IL-3 does not alter the characteristics of the [Ca2+]i that follows stimulation with C5a (12). Consequently, it appears that the ability of IL-3 to allow C5a to initiate LTC4 release results from its preconditioning of cPLA2, so that the brief transient [Ca2+]i that normally follows C5a stimulation is able to overlap with the pre-existing phosphorylated state of cPLA2 to allow its full activity (14).
It has been known for some time that the priming effect of IL-3 is apparent after overnight (18–24 h) treatment (11). Indeed, secretagogue-induced LTC4 release is often greater after 24 h of IL-3 treatment than after only 5–15 min of treatment. Unpublished functional studies have indicated that the priming effect is actually biphasic. A similar conclusion could be drawn from studies of the C5a-induced [Ca2+]i; as noted above, a short treatment does not alter the character of the response, while previous studies have also demonstrated a marked enhancement of the [Ca2+]i, characterized as a sustained elevation, occurring after 18–24 h of IL-3 incubation (11, 12). In preliminary results to be presented, we note that priming is not readily apparent after 2 h of priming, i.e., priming occurs after 5 min, disappears by 2 h, and returns by 24 h. The current studies explore the nature of the signaling events that lead to activation of cPLA2, comparing results for cells treated for 5–15 min to those after 18 h of treatment.
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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, cycloheximide, and Tris-HCl (Sigma, St. Louis, MO); crystallized human serum albumin (HSA; Miles Laboratories, Elkhart, IN); FCS and gentamicin (Life Technologies, Grand Island, NY); 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 (Roche, Indianapolis, IN); rabbit anti-ERK1 Ab and rabbit anti-ERK2 Ab (Santa Cruz Biotechnology, Santa Cruz, CA); rabbit anti-phospho-ERK (mitogen-activated protein kinase) Ab, phosphorylated and nonphosphorylated recombinant ERK2 proteins, and biotinylated m.w. markers (New England Biolabs, Beverly, MA); peroxidase-linked donkey anti-rabbit Ig Ab (Amersham, Arlington Heights, IL); PD98059 (Calbiochem, La Jolla, CA); recombinant human IL-3 (BioSource, Camarillo, CA); and fura-2/AM (Molecular Probes, Eugene, OR). 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).
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 (19, 20). Basophils and contaminating cells (lymphocytes and monocytes) that typically contaminate enriched basophil preparations expressed essentially equivalent levels of cPLA2 (14), ERK1, and ERK2 (15). In experiments involving the use of Western blot analysis of phosphorylated proteins or measurements of [Ca2+]i, basophil purities ranged from 80–99% to minimize the contribution from contaminating cells. The purity of basophils was determined by alcian blue staining (21).
Basophils are the major, if not sole, leukocytes to release histamine and to generate LTC4 in response to a combination of IL-3 and C5a, and IL-3 affects the basophil response directly (13). We also confirmed that basophils were the major source of LTC4 release stimulated by IL-3 (for 15 min or 18 h) plus C5a under our experimental conditions (see Results). Basophil preparations that were not as enriched as those used for phosphorylation and calcium studies were used for the experiments of histamine release and LTC4 generation (mean purity, 57 ± 6.3%; ranged from 15–96%).
Phosphorylation of ERKs and cPlA2.
The phosphorylation of ERKs was assessed using two different techniques: 1) phospho-ERK Ab (22), and 2) the electrophoretic mobility shift using anti-ERK1 and anti-ERK2 Abs (23, 24) as described previously (15). The phosphorylation of cPLA2 was assessed using the electrophoretic mobility shift with anti-cPLA2 Ab (14, 18). After basophils were incubated with or without IL-3 in RPMI 1640 containing 2% FCS and gentamicin (25 µg/ml) at 37°C in 5% CO2 incubator for the times indicated, reactions were stopped by adding ice-cold PAG (4-fold volume) and 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 number (1 x 106 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 5% 2-ME and subjected to 10% Tris glycine gel (NOVEX). After electrophoresis (160 V and 1.5 h for detection of ERKs phosphorylation, and 160 V and 3.5 h for cPLA2 phosphorylation), Gels were then transferred to supported nitrocellulose membranes (OPTITRAN, Schleicher & Schuell, Keene, NH) with a Trans Blot (NOVEX). The membranes were immersed overnight in Tris-buffered saline/Tween 20 containing 5% nonfat dry skim milk (Carnation, Los Angeles, CA). Immunoreactive proteins were detected using anti-phospho-ERK Ab, anti-ERK1 Ab, anti-ERK2 Ab, or anti-cPLA2 Ab, which were diluted in Tris-buffered saline/Tween 20 containing 1% skim milk for 4 h. After washing, the membranes were incubated for 1 h with HRP-conjugated anti-rabbit Ab. After washing, membrane-bound anti-rabbit Ig Ab was visualized with enhanced chemiluminescence Western blotting detection reagents (Amersham) and HyperECL luminescence detection film (Amersham).
[Ca2+]i measurements
Basophils were labeled with 1 µM fura-2/AM for 20 min at 37°C in RPMI 1640 containing 2% FCS (300,000–500,000 cells in 200 µl). After washing once with 200 µl of PAG, the cells were resuspended in PAG for loading in the microscope observation chamber (12, 14). Changes in [Ca2+]i were determined by digital video microscopy using techniques previously described in detail (12, 25). Briefly, 15 µl of cells (20,000–30,000) were loaded onto the siliconized coverslip of the microscope chamber and, after settling, overlaid with 1 ml of PAGCM buffer. After warming to 37°C, monitoring of the cells was begun, and after four frames (each frame is a single ratio measurement of a field of 30–100 cells) of prechallenge [Ca2+]i levels were acquired, the cells were challenged with 1 ml of stimulus in buffer. Data were then obtained for 50–150 frames at intervals of 1 to 10 s to determine the subsequent [Ca2+]i response.
LTC4 and histamine measurements
Fifty thousand basophils were stimulated in a final volume of 100 µl of RPMI 1640 containing 2% FCS and 25 µg/ml gentamicin at 37°C in 5% CO2 incubator. 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. RIA was performed using 100 µl of supernatant to determine LTC4 levels as previously described (1, 26). Supernatants (500 µl) were mixed with an equal volume of PAG buffer with 1.6% HClO4. After an overnight incubation at 4°C, the protein precipitate in each tube was pelleted by centrifugation (27), and the supernatants were assayed for histamine content by automated fluorometry (28). The percentage of total histamine release was calculated for the diluted supernatants after subtraction of spontaneous histamine release (29). Each condition tested was performed in duplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For most donor basophil preparations, C5a induces marked histamine
release from human basophils with little or no free
AA/LTC4 release (11, 12, 13). However,
the treatment with IL-3 for 15 min or overnight (18–24 h) enables C5a
to induce a significant amount of LTC4
(11, 12, 13). The kinetic characteristics of this priming
effect were examined. Basophils were treated with IL-3 for 15 min,
2 h, or 18 h before challenge with C5a. Supernatants were
harvested after 20 min of challenge, and LTC4 was
measured. As shown in Fig. 1
A,
treatment with IL-3 for 15 min significantly enhanced C5a-induced
LTC4 release; this enhancement was reduced to
levels similar to those observed for untreated cells by 2 h and
was markedly enhanced again by 18 h. This result suggested that
there are two distinct priming phases induced by IL-3. The supernatants
from these experiments were also examined for histamine release.
Similar to LTC4 release (but with far less
contrast), treatment with IL-3 for 15 min or 18 h enhanced
C5a-induced histamine release, because C5a is a strong secretagogue for
histamine release. Histamine release induced by C5a (no IL-3), IL-3 (15
min) plus C5a, IL-3 (2 h) plus C5a, and IL-3 (18 h) plus C5a was
50 ± 9, 70 ± 15, 58 ± 12, and 93 ± 7%,
respectively.
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The appearance of two phases in the kinetics of enhancement suggested
that the two phases resulted from two different mechanisms. We sought
out other indicators that these two phases of enhancement were
mechanistically different. Fig. 1
B demonstrates that the
concentration dependence of enhancement was different for the two
phases. The ED50 for acute priming (15 min) was
0.2 ng/ml (13.3 pM), a potency similar to that required for
phosphorylation of cPLA2 and to that for enhanced
free AA/LTC4 generation induced by low dose
ionomycin (0.1 µg/ml) (14). In contrast, the potency of
IL-3 for the late phase (18 h) of enhancement was much higher
(ED50 = >2 ng/ml; 133 pM).
The presence of a late priming effect that requires hours rather than
minutes suggested that protein synthesis may be required for this
phase. Thus, the effect of cycloheximide (protein synthesis inhibitor)
on both the acute (15 min) and late (18 h) priming by IL-3 was
examined. As shown in Fig. 2,
cycloheximide did not alter the effects of IL-3 on the C5a-induced
release of either LTC4 or histamine when cells
were pretreated for only 15 min. In contrast, the enhancement caused by
18 h of pretreatment with IL-3 was inhibited by cycloheximide;
complete inhibition occurred at 10 µM with an
ID50 of 0.3 µM. Both the enhancement of
LTC4 and histamine release were altered by the
inclusion of cylcoheximide, although reversal of the IL-3 effect was
more complete when examining LTC4 release. Cell
viability was examined by the trypan blue exclusion test; cell
viability was not affected by cycloheximide (results with no IL-3, with
IL-3 for 18 h, and with cycloheximide plus IL-3 for 18 h were
90.3 ± 5.5, 92.8 ± 4.8, and 91.3 ± 4.3%,
respectively; n = 3). These results suggested that the
effects of cycloheximide might not result solely from an effect on
viability. The observation that histamine release was the same in cells
treated with IL-3 and cycloheximide as in cells not treated with IL-3
also suggested that degranulation pathways remained functional.
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We have previously demonstrated that human basophils expressed
ERK1, ERK2, and cPLA2 (14, 15), and that
ERKs might regulate free AA for LTC4 generation
by phosphorylating cPLA2 following stimulation with
anti-IgE Ab or FMLP in human basophils (15). In
addition, phosphorylation of cPLA2 by IL-3 is
associated with increased free AA and LTC4
generation (14). However, a direct relationship between
activation of ERKs and of cPLA2 in response to IL-3
has not been reported yet. Phosphorylation of ERKs was assessed by two
different techniques: a change in the electrophoretic mobility could be
detected by anti-ERK1 and -2 Abs and changes in ERK phosphorylation
could be detected by immunoblotting with specific anti-phospho-ERKs
Ab, as previously described (15). Phosphorylated and
nonphosphorylated ERK2 proteins were used as a reference. As shown in
Fig. 3A, IL-3 induced
phosphorylation of ERKs at 15 min, and the phosphorylation returned
toward the basal level at 2 h. It was at basal levels at 18
h. The same results were obtained with the electrophoretic mobility
shift assay using anti-ERK1 Ab and anti-ERK2 Ab, respectively.
These immunoblottings by anti-ERK1 Ab and anti-ERK2 Ab also
demonstrated essentially equal protein loading for each lane, because
total protein mass from phosphorylated and nonphosphorylated forms were
the same (see Fig. 3A). Coinciding with phosphorylation of
ERKs, IL-3 also induced phosphorylation of cPLA2 at 15
min, and this phosphorylation decreased toward the basal level by
2 h and remained at basal levels at 18 h. These results
suggest a kinetic association between activation of ERKs and that of
cPLA2 in response to IL-3 in the context of the acute
priming effect. However, the priming effect after 18 h is not
explained by these temporal patterns of phosphorylation.
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It appears that the acute priming effect requires activation of the
ERK1/2 pathway. PD98059 is thought to be a specific inhibitor of
MEK1/2, the immediately antecedent kinases to the ERKs (15, 30, 31). This compound is often used to determine the involvement of
MEK/ERK activation. As shown in Fig. 3B, PD98059 at 100 µM
was capable of inhibiting the phosphorylation of both ERK1/2 and
cPLA2 when the cells were stimulated with IL-3,
suggesting that MEK/ERK regulates phosphorylation of
cPLA2.
Kinetics of phosphorylation of ERKs and of cPLA2, and LTC4 release induced by C5a after pretreatment with IL-3 (18 h)
C5a alone induces phosphorylation of cPLA2
(14) and ERKs (data not shown). However, the normal
C5a-mediated [Ca2+]i
response is rapid and transient and does not overlap with
phosphorylation of cPLA2 (12, 14).
Previous studies have also found that an 18-h pretreatment with IL-3
enhanced the C5a-induced
[Ca2+]i response by
specifically allowing the expression of a sustained
[Ca2+]i
(11). It therefore became of interest to examine the
characteristics of ERK and cPLA2 phosphorylation
following stimulation with C5a in cells that had been treated with IL-3
for 18 h. As noted above, in the presence of IL-3, phosphorylation
of both ERKs and cPLA2 had returned to resting levels
by 18 h. With this as a starting point, the phosphorylation of
these proteins was followed for the first 5 min following stimulation
with C5a. Modest phosphorylation of ERKs induced by C5a was observed at
30 s with a maximum phosphorylation from 1–5 min (Fig. 4A). Similar results were
obtained with electrophoretic mobility shift assay using anti-ERK1
Ab and anti-ERK2 Ab. These results also confirmed essentially equal
protein loading (data not shown). Phosphorylation of
cPLA2 followed phosphorylation of ERKs, with no
significant change at 30 s and a maximum change from 1–5 min
(Fig. 4A). A similar time course was observed for
C5a-stimulated cells that had been incubated for 18 h without IL-3
(data not shown), which is similar to the pattern observed in freshly
isolated cells as previously reported (14). In the cells
treated with IL-3 for 18 h, LTC4 release
followed phosphorylation of cPLA2 (Fig. 4B). It should be noted that the kinetics of
LTC4 release in these cells treated with IL-3 for
18 h were much slower (12 ± 0.5% of maximum release at 1
min and half-maximum at 3.5 min) than the release observed in cells
treated for only 15 min (12, 13), which was complete by
30–45 s. The rate of histamine release was similar for cells treated
for 15 min (13) and those treated for 18 h (86
± 5% of maximum at 30 s).
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As shown above, PD98059 inhibits the phosphorylation of ERK1/2 and
cPLA2 in response to IL-3 (Fig. 3B). We
have previously shown that it inhibits LTC4
release induced by FMLP and anti-IgE Ab (15). Thus,
the effect of PD98059 on the LTC4 release
enhanced by IL-3 (15 min or 18 h) was examined. FMLP-induced
LTC4 release was also examined as a control,
because PD98059 inhibits LTC4 release (but not
histamine release) induced by FMLP (15). As shown in Fig. 5, PD98059 inhibited
LTC4 release induced by IL-3 (15 min or 18
h) plus C5a as well as that by FMLP (no IL-3 pretreatment) at
comparable percent inhibition (the percent inhibitions of
LTC4 release induced with IL-3 (15 min) plus C5a,
IL-3 (18 h) plus C5a and FMLP by PD98059 were 64.1 ± 6.3,
78.0 ± 2.8, and 64.5 ± 8.5%, respectively). However,
histamine release induced by IL-3 (15 min or 18 h) plus C5a was
not affected by this compound (percent inhibitions of histamine release
induced with IL-3 (15 min) plus C5a and IL-3 (18 h) plus C5a by PD98059
were 11.5 ± 2.7 and 15 ± 2.5%, respectively;
n = 3). This result suggests that MEK-ERK activation is
essential for LTC4 release induced by IL-3 (15
min) plus C5a and that induced by IL-3 (18 h) plus C5a.
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B). Because
this compound requires a long preincubation (1 h) with the cells
(15, 22, 32, 33, 34, 35) and is not effectively washed from the
cells (data not shown), there was no method to isolate the actions of
the compound to the C5a-mediated pathway vs its effects on the
IL-3-induced pathway. For example, incubation of the cells with PD98059
for 1 h before a 15-min treatment with IL-3 and a subsequent
challenge with C5a significantly inhibited LTC4
release (Fig. 5A), but we could not distinguish between its
effects on the IL-3 vs C5a-mediated events in these acute priming
experiments. Therefore, the effects of this compound were tested on the
C5a-stimulated release of LTC4 in cells first
treated with IL-3 for 18 h. Cells were incubated for 18 h
with IL-3, and 100 µM PD98059 was added 1 h before challenge
with C5a. Under these conditions, PD98059 inhibited C5a-induced
LTC4 release (79.7 ± 10% inhibition
compared with cells not treated with PD98059), with no effect on
histamine release (n = 2). These results suggest that
the effects of PD98059 on C5a-induced LTC4
release from cells pretreated with IL-3 for 18 h mainly results
from inhibition of C5a-induced MEK/ERK activation, but not from that
induced by IL-3. Indeed, C5a-induced phosphorylation of ERK and
cPLA2 was also inhibited by PD98059 (data not
shown). Effect of EGTA or cycloheximide on [Ca2+]i and LTC4 release induced by C5a after pretreatment of IL-3 (18 h)
For cells incubated with IL-3 for 18 h, the data suggest that
the sustained [Ca2+]i is
the new element that allows C5a to induce LTC4
release (as will be discussed below). The kinetic characteristics of
the C5a-induced [Ca2+]i
in cells treated for 18 h with IL-3 are very similar to those of
the natural [Ca2+]i in
cells stimulated with FMLP (with sustained
[Ca2+]i)
(11). If EGTA is added with FMLP, the sustained phase of
[Ca2+]i (that dependent
on calcium influx) is ablated, and LTC4 release
is effectively inhibited, while histamine release remains largely
intact (11). However, these characteristics are different
from those of 15-min priming of IL-3 and stimulation by C5a.
C5a-induced LTC4 release after pretreatment with
IL-3 (15 min) are not affected by EGTA (12). We therefore
examined the effect of EGTA on LTC4 release in
cells treated with IL-3 for 18 h and stimulated with C5a. EGTA (2
mM) was simultaneously added with C5a, as performed previously
(12). The inclusion of EGTA completely inhibited
LTC4 release (LTC4 releases
without and with EGTA were 79 ± 50 and 4.5 ± 3.3
pmol/106 basophils, respectively; 95 ± 1%
inhibition by EGTA; n = 2). As expected, the
C5a-induced sustained phase of
[Ca2+]i was also
inhibited by EGTA, although the initial peak of
[Ca2+]i was not affected
(Fig. 6). Histamine release induced by
C5a was only marginally affected by EGTA (percent histamine release
without and with EGTA were 97 ± 4 and 80 ± 2%,
respectively; n = 2).
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), the sustained C5a-induced
[Ca2+]i after
pretreatment with IL-3 (18 h) was also inhibited by cycloheximide (Fig. 7). Taken together, these results suggest
that new protein synthesis might be required for the sustained
C5a-induced [Ca2+]i and
for the enhanced LTC4 release after treatment
with IL-3 for 18 h.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cPLA2 requires synchrony between its
phosphorylation (for enzymatic activity) and the increase in
[Ca2+]i (for its binding
to phospholipids) for its action to liberate AA (16, 17, 36, 37). In human basophils, cPLA2 is likely to
play a role in liberating AA for LTC4 synthesis
(as discussed below). In the absence of IL-3, C5a induces both
activation of ERK-cPLA2 and an increase in
[Ca2+]i (12, 14). However, the C5a-induced
[Ca2+]i response is
transient (<30–45 s) (12) on a time scale that does not
overlap with phosphorylation of cPLA2 (no
phosphorylation <30 s) (14), as portrayed in Fig. 8Ba. Because the translocation (or the binding) of
cPLA2 to the membrane is dependent on the level in
[Ca2+]i (16, 17, 36, 37), the transient C5a-induced association of
cPLA2 to the membranes may be dissociated (due to the
transient increase in
[Ca2+]i) before
phosphorylation of cPLA2. This separation in the times
between these two events explains the inability of C5a to induce free
AA/LTC4 generation (14). Although
both FMLP and C5a are pertussis toxin-sensitive stimuli in human
basophils (38), they differ in their characteristic
[Ca2+]i responses
(11, 12). In the absence of IL-3 treatment, the
FMLP-induced [Ca2+]i is
sustained and overlaps with the increase in cPLA2
phosphorylation (14). We postulate that the overlap in the
two events allows LTC4 release to follow
stimulation with FMLP, but not that with C5a. For basophils pretreated
with IL-3 for 5–15 min, synchrony between the two events is adjusted
by the fact that IL-3 induces phosphorylation of
cPLA2, so that the brief transient of
[Ca2+]i following C5a
overlaps in time with a phosphorylated cPLA2, allowing
free AA/LTC4 generation (14) (Fig. 8Bb). The generation of free AA/LTC4
is rapid and complete within 45 s, which is consistent with the
nature of a transient increase in
[Ca2+]i following C5a
stimulation (it returns to the basal level within 45 s) (12, 13). In the current studies we have shown that the overlap in
the two events was also adjusted by treatment of IL-3 for 18 h,
but by a different mechanism. After treatment with IL-3 for 18 h,
phosphorylation of cPLA2 had returned to basal levels
(as did ERK phosphorylation; Fig. 3A). However, this
treatment resulted in C5a inducing a sustained
[Ca2+]i, while the time
course of cPLA2 phosphorylation induced by C5a (Fig. 4A) was similar to that of cells not treated with IL-3
(14). The overlap in the two events occurred during the
sustained rise in
[Ca2+]i, much like the
response to FMLP in the absence of IL-3. Indeed, eliminating the second
phase of the [Ca2+]i,
which appears to result from an influx of extracellular calcium, by
inclusion of EGTA with C5a (Fig. 6), ablates LTC4
release in a manner similar to that found for FMLP (11).
The apparent dependence of LTC4 secretion (and
free AA generation, as shown previously (14)) on the
synchrony between these two events lends further support to the
conclusion that it is the activity of cPLA2 in human
basophils that is required for generating the free AA used for
LTC4 synthesis.
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B and data not shown). This compound also inhibited the
C5a-induced LTC4 release in IL-3-primed cells,
with both acute (15 min) and late (18 h) priming (Fig. 5), suggesting
that the activation of MEK/ERK is essential for
LTC4 generation induced by either acute priming
plus C5a or late priming plus C5a. However, we could not clearly
isolate and distinguish between two probable inhibitory events by this
compound, because this compound does not wash out of the cells (see
Results).
Treatment of the cells with cycloheximide also distinguished the two
forms of priming. Most notable was that cycloheximide effectively
inhibited the priming process that required overnight (18-h) incubation
with IL-3. However, this inhibition was restricted to functional
changes that were related to the late priming effects of IL-3.
C5a-mediated histamine release from cells treated with cycloheximide
and IL-3 was similar to release from cells not incubated with IL-3 for
18 h. Likewise, the
[Ca2+]i in cells treated
with IL-3 in the presence of cycloheximide resembled the normal
transient [Ca2+]i
observed in cells not treated with IL-3 (Fig. 7). In contrast,
cycloheximide did not inhibit priming that required only 15 min. The
sensitivity to cycloheximide of LTC4 release and
the enhanced [Ca2+]i
induced by treatment of IL-3 for 18 h suggest that this priming
process requires new protein synthesis.
The fact that both LTC4 release and the sustained
[Ca2+]i are sensitive to
cycloheximide (Figs. 2 and 7) and the apparent dependence of enhanced
LTC4 release on the sustained
[Ca2+]i (Fig. 6) suggest
that one important element of late priming is a change in the
regulation of cytosolic calcium levels during stimulation. Overnight
(18-h) priming with IL-3 also markedly enhances
[Ca2+]i following
anti-IgE Ab and FMLP, indicating that the alteration is a globally
effective change. It is curious, however, that cells that have not been
treated with IL-3 do have the means to sustain a cytosolic calcium
response; both FMLP and anti-IgE Ab induce reasonably sustained
[Ca2+]i (11, 41). Thus, IL-3 does not specifically alter the
[Ca2+]i by making
sustained responses possible; it appears to change the effectiveness of
whatever process allows a stimulus to induce an influx of extracellular
calcium. For C5a, a link to this influx process is normally deficient
for reasons not yet understood. IL-3 either up-regulates some
component(s) of the influx pathway, such that even C5a is capable of
using it, or IL-3 also enables the linkage for C5a to use this normally
present pathway. Until more is understood about the regulation of the
[Ca2+]i, it is not
possible to discriminate among various alternatives. It should also be
noted that while the purity of the basophils for many of these
experiments was very high, and we did not detect a dependence of the
priming effects on basophil purity, it remains possible that priming
could result from an indirect effect of IL-3 on the remaining
contaminating cells in these preparations.
In conclusion, the mechanistic characteristics of late priming by IL-3 differ from those of the acute effect. The acute effect by IL-3, but not the late effect, is associated with ERK-cPLA2 activation induced by IL-3. However, the late effect is related to an enhancement of the C5a-induced [Ca2+]i response, and the late priming process appears to require protein synthesis.
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Acknowledgments |
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Footnotes |
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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; ERK, extracellular signal-regulated kinase; AA, arachidonic acid; HSA, human serum albumin; PAG, PIPES-albumin-glucose; PAGCM, PAG supplemented with 1 mM CaCl2 and 1 mM MgCl2; MEK, mitogen-activated protein/ERK kinase; cPLA2, cytosolic phospholipase A2; [Ca2+]i, cytosolic calcium response; C5a, anaphylatoxin split product of C component; fura-2/AM, fura-2-acetoxymethyl ester.
Received for publication September 24, 1999. Accepted for publication January 11, 2000.
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) or medium control
(
) for 20 min. B, Basophils were incubated with IL-3
at the concentrations indicated for 15 min (circles) or 18 h
(square), and were stimulated by C5a (50 ng/ml; filled symbols) or
medium control (open symbols) for 20 min. Reactions were terminated by
the addition of ice-cold PAG-EDTA, and the cells were centrifuged.
Supernatants were collected and subjected to LTC4 assay.
Values are the mean ± SEM of four experiments (A)
and the mean ± range of two experiments (B).
Control LTC4 release induced by IL-3 (10 ng/ml, 15 min)
plus C5a and by IL-3 (10 ng/ml, 18 h) plus C5a were 29 ± 19
and 53 ± 30 pmol/106 basophils, respectively
(B).
) or 18 h (open column) and were stimulated by C5a
(50 ng/ml) for 20 min. Reactions were terminated by the addition of
ice-cold PAG-EDTA, and the cells were centrifuged. Supernatants were
collected and subjected to LTC4 (A) and
histamine (B) assay. Values are the mean ± SEM of
three experiments. LTC4 releases induced by IL-3 (15 min)
plus C5a and by IL-3 (18 h) plus C5a were 56 ± 8 and 89 ±
49 pmol/106 basophils, respectively. The percent histamine
releases induced by IL-3 (15 min) plus C5a and by IL-3 (18 h) plus C5a
were 72 ± 10% and 88 ± 3%, respectively.
