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 ~10nM. 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.

Figure 1. PCI-32765 inhibits IgE-dependent functions

Panel A: Purified basophils were stimulated with 0.5 µg/ml of monoclonal anti-IgE antibody for 30 minutes (n=4). A portion of the samples were immediately fixed and stored for analysis by flow cytometry for CD63 (), CD203c (), or CD11b (). The results were calculated from the net mean fluorescence intensity (above non -stimulated controls). A portion of the supernatants were analyzed for histamine (○) or LTC4 (□) by automated flourometry or radioimmunoassay, respectively. Histamine release following challenge with anti-IgE in the presence of vehicle control (0.0005% DMSO) averaged 45±17% and LTC4 release averaged 57±3 ng/µg histamine. In parallel, btk occupancy by the inhibitor was analyzed by a fluorescent probe assay ()(n=2). Panel B: IL-4 and histamine secretion in basophil-enriched (filled symbols) and purified basophil (open symbols) suspensions. Cells were incubated in media for 4 hours and stimulated with 20 ng/ml anti-IgE to optimize IL-4 secretion. Values are the mean±SEM of 3 experiments for the basophil enriched suspensions and 2 experiments (mean±range) for purified suspensions. Histamine release following challenge with anti-IgE in the presence of vehicle control (a maximum of 0.004% DMSO) from enriched suspensions (■) averaged 50±12% and IL-4 secretion (●) in enriched suspensions averaged 215±67 pg/10⁶ basophils, similarly histamine release (□) in purified suspensions averaged 35±4% and control IL-4 secretion (○) in purified suspensions averaged 31±2 pg/10⁶ basophils.

Figure 2. PCI-32765 does not inhibit non-IgE-dependent functions

Panel A: Purified basophils were stimulated with 100 nM FMLP or 100 ng/ml C5a in the presence or absence of 50 nM PCI -32765 (n=4). Histamine release in stimulated vehicle control (0.0005% DMSO) samples averaged 92±7% for FMLP and 100±5% for C5a, LTC4 release in stimulated vehicle controls samples average d 132±38 ng/µg histamine for FMLP and 17±3 ng/µgH for C5a. Statistical analysis showed all differences were p>0.05 (H₀ = 1.0). Panel B: Purified basophils (n=2) were stimulated with 1 µM FMLP (○) or 0.5 µg/ml A23187 (■,●) and supernatants analyzed for histamine (●) or IL-4 secretion (■). Histamine release in vehicle control (a maximum of 0.1% DMSO) samples averaged 64±22% for A23187 and 44±13% for FMLP and IL-4 release averaged 305±38 pg/10⁶ basophils for A23187. FMLP did not induce IL-4 release.

Figure 3. PCI-32765 does not inhibit IL-3-induced IL-13 secretion by human basophils

Purified basophils were cultured in media for 18 hours with 10 ng/ml IL-3 and PCI-32765 at the concentrations shown. Supernatants were harvested after 18h incubation and assayed for IL-13 by ELISA. Shown are the mean±SEM values (n=3), statistical analysis showed all differences were p>0.05. There was no IL-13 measured in cultures without IL-3 present.

Figure 4. Partial inhibition of the IgE-mediated increase in cytosolic calcium by PCI-32765

Fura-2 loaded purified basophils were incubated with vehicle control (0.0005% DMSO) or 50 nM PCI-32765 for 10 minutes prior to the addition of 0.5 µg/ml anti-IgE antibody and the cytosolic calcium response monitored. The 350/380 excitation ratio is plotted for the average of two experiments.

Figure 5. PCI-32765 does not inhibit IgE-mediated down-regulation of syk

Purified basophils were incubated in media with the concentrations of PCI-32765 shown or DMSO vehicle (0.0005%) for 10 minutes prior to the addition of 0.5 µg/ml anti-IgE antibody. The samples were then incubated for 18 hours prior to lysis of cell pellets for Western blotting for syk and p85 (which acts as the loading control). The experiment represents one of two experiments using different basophil preparations.