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Expression of FcεRI on basophils and mast cells is modulated by IgE Ab. Previous studies have noted in vivo receptor expression dynamics that are discordant with expectations derived from in vitro studies. The current study presents a formal hypothesis to explain the discordant observations and tests two assumptions that underlie a proposed model of receptor dynamics. After first showing that a murine model of receptor expression on basophils recapitulates observations made using human basophils, the effect of changes in IgE on basophil egress rates was examined. In the proposed model, egress rates from bone marrow (BM) were assumed to be unaffected by changes in IgE concentration. Egress was tested by examining the labeling of BM and peripheral blood (BL) basophils at various times after injection of BrdU with and without injection with IgE. The IgE Ab did not alter the appearance of BrdU label in peripheral BL basophils. In addition, BM and BL basophils were responsive to the elevations in IgE, with receptor expression increasing on BM basophils before BL basophils. It was also noted that BL basophils expressed ∼50% of the receptor density of BM basophils. There was a 3-fold greater synthetic rate of FcεRI on BM basophils that readily explained the difference. These results provide support for the proposed hypothesis of rapid changes in receptor expression being controlled by cell replacement. The studies also support a model whereby receptor expression is limited by cell division and that basophils, once mature, slow their rate of receptor synthesis.

IL-3 is a potent priming cytokine for human basophils, inducing an increase of mediator release after stimulation. The mechanism of IL-3 priming of the basophil response to FcεRI aggregating stimuli remains unknown. We explored the regulation of several elements of IgE-mediated signaling by a short priming with IL-3. Early signaling events such as phosphorylation of Syk, Shc, linker for activation of T cells, and the calcium signal were not statistically affected by acute IL-3 priming. Downstream in the signaling cascade, a point of up-regulation was found at the level of Raf-1-Mek-Erk. Although the phosphorylation of Raf-1 was not changed by IL-3 priming, IL-3-primed anti-IgE-stimulated basophils showed a strong synergism for Mek and Erk phosphorylation when compared with either IL-3 or anti-IgE alone; pre-exposure to IL-3 induced a final 13-fold average increase over anti-IgE-induced Erk phosphorylation (6-fold above the sum of anti-IgE and IL-3 alone). The kinetics, dose response, and pharmacologic characteristics of the IL-3 priming of stimulus-induced Erk phosphorylation support the involvement of a yet unknown mechanism that is independent of IL-3-induced Erk and PI3K activation. This type of preactivation can be mimicked by incubation with the Ser-Thr kinase inhibitors, Ro-81–3220, or bisindoylmaleimide II.

Previous studies have indicated a redundancy in the effects of the cytokines, IL-3, IL-5, and nerve growth factor (NGF) on acute priming of human basophils. In the current study, we have examined the effects of these three cytokines on 18-h priming for leukotriene C4 generation, their ability to induce FcεRIβ mRNA expression, or their ability to sustain basophil viability in culture. We also examine a variety of the signaling steps that accompany activation with these cytokines. In contrast with the ability of IL-3 to alter secretagogue-mediated cytosolic calcium responses following 18-h cultures, 18-h treatment with IL-5 or NGF did not affect C5a-induced leukotriene C4 generation or alter C5a-induced intracellular Ca2+ concentration elevations. IL-3 and IL-5, but not NGF, induced FcεRIβ mRNA expression and all three improved basophil viability in culture with a ranking of IL-3 > IL-5 ≥ NGF. All three cytokines acutely activated the extracellular signal-regulated kinase pathway and the signaling elements that preceded extracellular signal-regulated kinase and cytosolic phospholipase A2 phosphorylation, consistent with their redundant ability to acutely prime basophils. However, only IL-3 and IL-5 induced Janus kinase 2 and STAT5 phosphorylation. This pattern of signal element activation among the three cytokines most closely matched their ability to induce expression of FcεRIβ mRNA. Induction of the sustained calcium signaling that follows overnight priming with IL-3 appeared to be related to the strength of the early signals activated by these cytokines but the relevant pathway required was not identified. None of the signaling patterns matched the ability of the cytokines to promote basophil survival.

Although FcεR have been detected on human eosinophils, levels varied from moderate to extremely low or undetectable depending on the donor and methods used. We have attempted to resolve the conflicting data by measuring levels of IgE, FcεRI, and FcεRII in or on human eosinophils from a variety of donors (n = 26) and late-phase bronchoalveolar lavage fluids (n = 5). Our results demonstrated little or no cell surface IgE or IgE receptors as analyzed by immunofluorescence and flow cytometry. Culture of eosinophils for up to 11 days in the presence or absence of IgE and/or IL-4 (conditions that enhance FcεR on other cells) failed to induce any detectable surface FcεR. However, immunoprecipitation and Western blot analysis of eosinophil lysates using mAb specific for FcεRIα showed a distinct band of approximately 50 kDa, similar to that found in basophils. Western blotting also showed the presence of FcR γ-chain, but no FcεRIβ. Surface biotinylation followed by immunoprecipitation again failed to detect surface FcεRIα, although surface FcRγ was easily detected. Since we were able to detect intracellular FcεRIα, we examined its release from eosinophils. Immunoprecipitation and Western blotting demonstrated the release of FcεRIα into the supernatant of cultured eosinophils, peaking at approximately 48 h. We conclude that eosinophils possess a sizable intracellular pool of FcεRIα that is available for release, with undetectable surface levels in a variety of subjects, including those with eosinophilia and elevated serum IgE. The biological relevance of this soluble form of FcεRIα remains to be determined.

Human basophils secrete histamine and leukotriene C4 (LTC4) in response to various stimuli, such as Ag and the bacterial product, FMLP. IgE-mediated stimulation also results in IL-4 secretion. However, the mechanisms of these three classes of secretion are unknown in human basophils. The activation of extracellular signal-regulated kinases (ERKs; ERK-1 and ERK-2) during IgE- and FMLP-mediated stimulation of human basophils was examined. Following FMLP stimulation, histamine release preceded phosphorylation of ERKs, whereas phosphorylation of cytosolic phospholipase A2 (cPLA2), and arachidonic acid (AA) and LTC4 release followed phosphorylation of ERKs. The phosphorylation of ERKs was transient, decreasing to baseline levels after 15 min. PD98059 (MEK inhibitor) inhibited the phosphorylation of ERKs and cPLA2 without inhibition of several other tyrosine phosphorylation events, including phosphorylation of p38 MAPK. PD98059 also inhibited LTC4 generation (IC50 = ∼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.

Human basophils are an important source of IL-4, a cytokine that is central to the pathogenesis of allergic inflammation. Recent reports have indicated that these cells also generate IL-13, which shares a number of biologic properties with IL-4. We found basophils to be the major source of IL-13 produced in mixed leukocyte cultures following 20-h activation with a variety of stimuli. While the magnitude of IL-4 protein generated correlated with the percent histamine secreted (r = 0.8; p = 0.007), there was no relationship between the levels of IL-13 detected and the amount of either IL-4 or histamine in cultures activated with IL-3/anti-IgE. The induction of IL-13 secretion also occurred in response to IL-3 alone, without concomitant secretion of either IL-4 or histamine. Although previously shown to inhibit IL-4 secretion, the phorbol ester PMA was a potent stimulus for IL-13 generation from basophils, and this secretion was sensitive to the protein kinase C inhibitor, bisindolylmaleimide. In contrast, bisindolylmaleimide did not prevent cytokine secretion induced by either anti-IgE or IL-3. The immunosuppressant, FK506, while strikingly inhibiting the accumulation of IL-4 mRNA and the secretion of protein in response to IL-3/anti-IgE, had no effect on the generation of IL-13 in these cultures; the resistance was attributed to the IL-3-dependent signaling. Similarly, FK506 had no effect on the secretion of IL-13 in basophil cultures stimulated with PMA. This study suggests that multiple intracellular mechanisms control the generation of IL-13 in basophils, some of which are distinct from those regulating IL-4.