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 Fc(epsilon)RI 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.


CONCLUSIONS
Taken together, these data suggest that the hyperreleasability of IgE(+) donors is associated with low levels of SHIP and implicate SHIP as an additional regulator of secretion in human basophils.
Vonakis
B M
BM
Division of Allergy and Clinical Immunology, The Johns Hopkins School of Medicine, Baltimore, MD, USA.
Gibbons
S
S
Sora
R
R
Langdon
J M
JM
MacDonald
S M
SM
D001491:Basophils / Q000187:drug effects / Q000201:enzymology / Q000276:immunology*; D014408:Biomarkers, Tumor; D002478:Cells, Cultured; D004792:Enzyme Precursors / Q000378:metabolism; D006636:Histamine Release; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000276:immunology*; D007073:Immunoglobulin E / Q000276:immunology; D047908:Intracellular Signaling Peptides and Proteins; D008214:Lymphocytes / Q000378:metabolism; D008222:Lymphokines / Q000494:pharmacology*; D009000:Monocytes / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000502:physiology; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology; D015398:Signal Transduction; D000072377:Syk Kinase; D000090324:Tumor Protein, Translationally-Controlled 1; D019061:src-Family Kinases / Q000378:metabolism
D014408:Biomarkers, Tumor; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D008222:Lymphokines; D018129:Phosphatidylinositol Phosphates; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000090324:Tumor Protein, Translationally-Controlled 1; C060974:phosphatidylinositol 3,4,5-triphosphate; D007073:Immunoglobulin E; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; C000606897:Syk protein, mouse; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D010744:Phosphoric Monoester Hydrolases; C098706:INPPL1 protein, human; D000072183:Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases