Within the general population, individuals can be found whose basophils do not secrete after stimulation through the immunoglobulin (Ig) E receptor. In this study we compared two groups of donors, those whose basophils responded with 65+/-16% histamine release to an optimal concentration of anti-IgE antibody and those whose basophil response was not statistically different from nonstimulated release (1+/-1%). We show that these so-called nonreleasing basophils have at least 10-fold lower expression of the tyrosine kinases, lyn and syk, but normal expression of the tyrosine kinase Btk when compared with the panel of releasing basophils. Indeed, maximum histamine release correlated with expression of both syk (Spearman rank correlation coefficient [Rs] = 0.98) and lyn (Rs = 0.93). In contrast, equivalent levels of messenger RNA (mRNA) for lyn and syk kinase were found for both groups. By sequencing a critical region in the syk mRNA, our results also demonstrate that the frame shift mutation in syk leading to a premature stop codon which has been observed in other cell types is not present in nonreleasing human basophils. Our results suggest that there may be translational or post-translational regulatory mechanisms specific to the expression of two important FcepsilonRI-associated signaling elements in basophils.
Mediator release from human basophils is a self-limited process, but down-regulation of the signaling cascades leading to secretion of leukotriene C4 (LTC4) is controlled independently of the pathway leading to IL-4 secretion. In the current studies, we have explored the regulation of upstream signaling events leading to activation of extracellular signal-related kinases (ERKs; previously shown to be required for LTC4 generation) in human basophils. IgE-, but not FMLP-mediated activation, induced sustained tyrosine phosphorylation of syk, of shc, and an association of shc to the Grb2/son of sevenless 2 complex. In contrast, IgE-mediated activation resulted in transient activation of p21ras and mitogen-activated protein/ERK kinase 1, which were kinetically associated with phosphorylation of ERKs. The canonical Shc/Grb2/son of sevenless pathway to activation of p21ras is therefore sustained, while p21ras activity is not. We have previously shown that phosphatidylinositol 3 kinase activity is required for p21ras activity and, in the current studies, we show that of the p85-sensitive forms of p110 possible, basophils express only p110 δ and that there are no changes in association between p21ras and p110 δ in stimulated basophils. We used the generation of phospho-Akt as a marker of the presence of phosphatidylinositol-3,4,5-trisphosphate and found that phospho-Akt is transient on a time scale consistent with p21ras activity. On the basis of information obtained in these and other studies, we localize down-regulation of IgE-mediated LTC4 secretion to a region of the signaling cascade antecedent to p21ras activation, downstream of phosphatidylinositol 3 kinase activity and probably involving regulation of phosphatidylinositol-3,4,5-trisphosphate levels.
Secretion from mast cells and basophils, two cells central to immediate hypersensitivity reactions, has characteristics that suggest the existence of intrinsic signal transduction processes that limit the extent of the cell's response. This process(es) has been termed desensitization. One goal of current research efforts is to determine the mechanisms used by mast cells and basophils to down-regulate an ongoing secretory reaction. Recent studies have indicated that, like secretion itself, the mechanisms of down-regulation or desensitization differ according to the mediator being studied. Thus, for human basophils, there appear to be distinct signaling pathways leading to the secretion of the three major classes of mediators--granules contents, lipids, and cytokines--and each pathway appears to have distinct down-regulatory processes. For an ongoing secretory reaction, the secretion of histamine and LTC4 are limited by a process that does not involve the earliest steps in activation, activation of the early tyrosine kinases, lyn and syk. These early events persist for long periods which more appropriately correspond to the regulation of cytokine secretion. Recent studies have also indicated that the process of desensitization is altered during stimulation in the absence of extracellular calcium, the traditional method of examining this process. These studies indicate that down-regulation studied in this manner is not dependent on any of the signaling events currently defined as being necessary for secretion. A variety of processes are discussed and potential mechanisms based on most recent studies using cell lines are explored.
Taken together with published studies using other cell types, we conclude that piceatannol does not inhibit secretion from human basophils by inhibiting the activity of syk kinase.
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