Inhibition of IgE-mediated secretion from human basophils with a highly selective Bruton's tyrosine kinase, Btk, inhibitor

Abstract

The study of receptor-mediated signaling in human basophils is often limited by the availability of selective pharmacological agents. The early signaling reaction mediated by FcεRI aggregation is thought to require the activity of Bruton's tyrosine kinase (btk), an enzyme that has been identified as important in B cells signaling because mutations lead to X-linked agammaglobulinemia. This study uses the btk selective irreversible inhibitor, PCI-32765, to explore the role of btk in a variety of functions associated with the activation of human basophils. Nine endpoints of basophil activation were examined: induced cell surface expression of CD63, CD203c, CD11b; induced secretion of histamine, LTC4, IL-4 and IL-13; the cytosolic calcium response; and the induced loss of syk kinase. Four stimuli were examined; anti-IgE antibody, formyl-met-leu-phe (FMLP), C5a and IL-3. For stimulation with anti-IgE, PCI-32765 inhibited CD63, histamine, LTC4 and IL-4 secretion with an IC50 of 3–6 nM (with 100% inhibition at 50 nM) and it inhibited CD203c and CD11b and the cytosolic calcium response with and IC50 of 30–40 nM. Fifty percent occupancy of btk with PCI-32765 occurred at ~ 10 nM. Consistent with btk functioning downstream or in parallel to syk activation, PCI-32765 did not inhibit the loss of syk induced by anti-IgE in overnight cultures. Finally, PCI-32765 did not significantly inhibit basophil activation by FMLP or C5a and did not inhibit IL-13 release induced by IL-3. These results suggest that btk is specifically required for IgE-mediated activation of human basophils.

Introduction

During IgE-mediated secretion in basophils and mast cells, there are several kinases that have been identified as being critical for the initiation of the early reaction. For example, syk, a tyrosine kinase that is related to ZAP-70, is a non-redundant kinase in basophils and mast cells whose activity determines a very large number of downstream events following the aggregation of FcεRI. Pharmacological agents that effectively ablate syk's kinase activities eliminate all mediator release [1]. Likewise, complete inhibition of PI3 kinase, and in particular, PI3 kinase delta, also completely blocks all mediator secretion [2], [3]. The kinase btk has a well defined role downstream of the B cell receptor; human mutations that inactivate Btk prevent B cell maturation and cause X-linked agammaglobulinemia. Btk has also been shown to be important in IgE-mediated signaling using rodent mast cell models [4], [5]. However, the tools for clearly determining Btk's relevance to secretion in human basophils and mast cells have not been previously available. For example, dasatinib is an inhibitor that targets several tyrosine kinases. This drug is also a potent and efficacious inhibitor of btk and has been used to study human basophils [6]. However, because dasatinib inhibits a relatively broad range of kinases [7], effects of this inhibitor do not address the specific role of Btk in basophils. Evidence indicates that recruitment of btk to the plasma membrane results from the generation of phosphotidylinositol 3,4,5, trisphosphate (PIP3) by PI3 kinase [8]. In some cells, recruited btk is responsible for the phosphorylation and activation of PLC-γ1/2 which is, in turn, responsible for the generation of inositol, 3,4,5 triphosphate (IP3) and the initiation of the elevation in cytosolic calcium [9]. In human basophils, inhibition of PI3 kinase has only a modest effect on the IgE-mediated cytosolic calcium response [3] so it is unclear whether there is, in fact, an essential role for btk in modulating human basophil function. In recent years, a selective irreversible inhibitor of human btk, PCI-32765, has been developed [10], [11]. This study tests whether btk activity is critical for the expression of multiple indicators of basophil activation in response to a range of physiological stimuli.