Dual Phase Priming by IL-3 for Leukotriene C₄ Generation in Human Basophils: Difference in Characteristics Between Acute and Late Priming Effects

Materials

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 cPlA_2.

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 × 10⁶ cell equivalents/lane) were diluted with an equal volume of 2× 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.

Biphasic characteristics of priming by IL-3

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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FIGURE 1.

A, The release of LTC₄ induced by C5a after pretreatment with IL-3 for different times. B, Dose-dependent effect of pretreatment with IL-3 (15 min or 18 h) on C5a-induced release of LTC₄. A, Basophils were incubated without (time 0) or with IL-3 (10 ng/ml) for the times indicated and were stimulated by C5a (50 ng/ml; ▪) 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).

Kurimoto et al. (13) previously demonstrated that basophils are the major, if not sole, leukocytes to generate LTC₄ in response to a combination of IL-3 (early effects) and C5a, and IL-3 affects the basophil response directly. To confirm these results under our experimental conditions we also examined LTC₄ release from pure (70–96%) and impure (6%) basophil suspensions (from the same donor preparations) in response to combinations of IL-3 (15 min or 18 h) and C5a. From cells treated for either 15 min or 18 h, LTC₄ release was proportional to the basophil purity (data not shown), indicating that basophils are the major leukocytes to generate LTC₄ in response to a combination of IL-3 and C5a.

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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FIGURE 2.

The effect of cycloheximide on LTC₄ release (A) and histamine release (B) induced by C5a after pretreatment with IL-3 (15 min or 18 h). Basophils were incubated without or with cycloheximide at the concentrations indicated for 15 min. The cells were incubated with or without IL-3 (10 ng/ml) for 15 min (▨) 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.

Phosphorylation of ERKs (ERK1 and ERK2) and of cPLA₂ induced by IL-3 in human basophils

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. 3⇓A, 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. 3⇓A). 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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FIGURE 3.

A, Kinetics of phosphorylation of ERKs and of cPLA₂ induced by IL-3 in human basophils. Basophils were incubated without (time 0) or with IL-3 (10 ng/ml) for the times indicated. Reactions were stopped with the addition of ice-cold PAG, and the cells were microfuged. Cell pellets were lysed and subjected to Western blot analysis as described in Materials and Methods. The Western blot shown is representative of three separate experiments. B, Effect of PD98059 on IL-3-induced phosphorylation of ERKs and cPLA₂. Basophils were preincubated with PD98059 (100 μM) or DMSO control for 1 h and were stimulated by IL-3 for 5 min. Cell pellets were lysed and subjected to Western blot analysis as described in Materials and Methods. The Western blot shown is representative of two separate experiments.

Previous studies noted that the full expression of the acute priming effect of IL-3 required 5 min of pretreatment (13), consistent with the time period that is required for maximum phosphorylation of cPLA₂ induced by IL-3 (14). We therefore examined the IL-3-induced phosphorylation of ERKs within this short time frame. Phosphorylation of ERKs was not detected at 1 min, but significant phosphorylation was observed from 5–15 min (data not shown). A similar kinetic pattern was observed for phosphorylation of cPLA₂ in response to IL-3 (14). These results also suggested the kinetic association of activation of ERKs with that of cPLA₂ and the acute priming effect of IL-3 for LTC₄ release.

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. 3⇑B, 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. 4⇓A). 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. 4⇓A). 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. 4⇓B). 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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FIGURE 4.

A, Kinetics of phosphorylation of ERKs and cPLA₂ induced by C5a after pretreatment with IL-3 (18 h). B, Kinetics of release of LTC₄ and histamine induced by C5a after pretreatment with IL-3 (18 h). A, The cells were incubated with IL-3 (10 ng/ml) for 18 h and stimulated with C5a (50 ng/ml) for the times indicated. Reactions were stopped by the addition of ice-cold PAG, and the cells were microfuged. Cell pellets were lysed and subjected to Western blot analysis as described in Materials and Methods. The Western blot shown is representative of two separate experiments. B, Basophils were incubated with IL-3 (10 ng/ml) for 18 h and stimulated by C5a (50 ng/ml) for the times indicated. LTC₄ (□) and histamine (•) were measured as described in Materials and Methods. Values are the mean ± range of two experiments. The percent histamine release and the LTC₄ release at 20 min were 93 ± 7 and 58 ± 11 pmol/10⁶ basophils, respectively.

Effect of PD98059 on C5a-induced LTC₄ release after pretreatment of IL-3 for 15 min or 18 h

As shown above, PD98059 inhibits the phosphorylation of ERK1/2 and cPLA₂ in response to IL-3 (Fig. 3⇑B). 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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FIGURE 5.

The effect of PD98059 on LTC₄ release induced by C5a after pretreatment with IL-3 (15 min or 18 h). Basophils were incubated with or without PD98059 (100 μM) for 1 h. The cells were further incubated with or without IL-3 (10 ng/ml) for 15 min and stimulated by C5a (50 ng/ml) or FMLP (1 μM) for 20 min (A). Basophils were incubated with or without PD98059 (100 μM) for 1 h. The cells were further incubated with or without IL-3 (10 ng/ml) for 18 h and stimulated by C5a for 20 min (B). 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 three experiments for IL-3 plus C5a and the mean ± range of two experiments for FMLP. LTC₄ releases induced by IL-3 (15 min) plus C5a, FMLP (alone), and IL-3 (18 h) plus C5a were 31 ± 11, 53 ± 29, and 40 ± 15 pmol/10⁶ basophils, respectively. Asterisks indicate a statistically significant difference from LTC₄ release induced by IL-3 (15 min or 18 h) plus C5a (p < 0.05, as determined by paired t test).

We considered the possibility that LTC₄ release stimulated by C5a in IL-3-pretreated cells might not depend on a pathway similar to FMLP in cells not treated with IL-3. As noted above, PD98059 effectively inhibited the phosphorylation of ERK1/2 and cPLA₂ induced by IL-3 (Fig. 3⇑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. 5⇑A), 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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FIGURE 6.

The effect of EGTA on the [Ca²⁺]_i response induced by C5a after pretreatment with IL-3 (18 h). Basophils were incubated with IL-3 (10 ng/ml) for 18 h, washed, and labeled with fura-2/AM. After washing, the cells were stimulated with C5a (50 ng/ml) in the presence or the absence of 2 mM EGTA (added with the stimulus). The [Ca²⁺]_i response was analyzed as described in Materials and Methods. The result shown is representative of two separate experiments.

We then examined the effect of cycloheximide on the sustained C5a-induced [Ca²⁺]_i after pretreatment with IL-3 for 18 h. Consistent with results for LTC₄ release (Fig. 2⇑), 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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FIGURE 7.

The effect of cycloheximide on the [Ca²⁺]_i response induced by C5a after pretreatment with IL-3 (18 h). Basophils were incubated for 18 h without IL-3 (A), with 10 ng/ml IL-3 (B), or with IL-3 plus 10 μM cycloheximide (C). The cells were then washed and labeled with fura-2/AM. After washing, the cells were stimulated with C5a (50 ng/ml). The [Ca²⁺]_i response was analyzed as described in Materials and Methods. The results shown are the average of two separate experiments.