Src homology 2 domain-containing inositol 5′ phosphatase is negatively associated with histamine release to human recombinant histamine-releasing factor in human basophils

Background: The human recombinant histamine-releasing factor (HrHRF) acts as a complete stimulus for histamine release and IL-4 secretion from a subpopulation of highly allergic donor basophils, termed IgE⁺ basophils. Additionally, IgE⁺ basophils release histamine to other secretogues, IL-3, and deuterium oxide. We hypothesized that IgE⁺ basophils were hyperreleasable. Objective: Deficiencies in early signal transduction events associated with FcεRI lead to a nonreleasable phenotype, whereas the Src homology 2 domain-containing inositol 5' phosphatase (SHIP) knockout mice have hyperreleasable mast cells. The purpose of this study was to ascertain whether a difference in intracellular signaling molecules could explain the hyperreleasable phenotype of human IgE⁺ basophils. Methods: Basophils were purified by means of double Percoll gradients and negative selection with magnetic beads. Cell lysates were Western blotted for the tyrosine kinases Lyn and Syk and the phosphatase SHIP. Additionally, histamine release to HrHRF was performed in addition to real-time RT-PCR to investigate mRNA for SHIP. Results: We show a striking negative correlation between the amount of SHIP protein per cell equivalent, but not Lyn or Syk, and maximum histamine release to HrHRF. This deficiency of SHIP was observed in basophils, but not lymphocytes or monocytes, of these IgE⁺ donors. Additionally, levels of mRNA for SHIP did not differ between IgE⁺ and IgE^- donor basophils, which is consistent with a posttranscriptional mechanism of protein regulation. SHIP and phosphatidylinositol 3-kinase reciprocally regulate phosphatidylinositol (3,4,5) triphosphate levels. We also demonstrated that Ly294002, the phosphatidylinositol 3 kinase inhibitor, prevented HrHRF-induced histamine release in IgE⁺ donor basophils. Conclusion: Taken together, these data suggest that the hyper-releasability of IgE⁺ donors is associated with low levels of SHIP and implicate SHIP as an additional regulator of secretion in human basophils.