Inhibition of IgE-mediated secretion from human basophils with a highly selective Bruton's tyrosine kinase, Btk, inhibitor.
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.
Johns Hopkins University, Asthma and Allergy Center, Baltimore, MD 21224, USA. dmacglas@jhmi.edu
Donald
D
MacGlashan
Lee A
LA
Honigberg
Ashley
A
Smith
Joseph
J
Buggy
John T
JT
Schroeder
D000225:Adenine / Q000031:analogs & derivatives; D000077329:Agammaglobulinaemia Tyrosine Kinase; D015703:Antigens, CD / Q000096:biosynthesis / Q000276:immunology; D001491:Basophils / Q000187:drug effects / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D005434:Flow Cytometry; D006636:Histamine Release / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007073:Immunoglobulin E / Q000276:immunology*; D018793:Interleukin-13 / Q000378:metabolism; D015847:Interleukin-4 / Q000378:metabolism; D010880:Piperidines; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D011720:Pyrazoles / Q000494:pharmacology*; D011743:Pyrimidines / Q000494:pharmacology*
D015703:Antigens, CD; D018793:Interleukin-13; D010880:Piperidines; D011720:Pyrazoles; D011743:Pyrimidines; C551803:ibrutinib; D015847:Interleukin-4; D007073:Immunoglobulin E; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D000225:Adenine; D002118:Calcium
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