The src homology 2-containing inositol phosphatase (SHIP) is the gatekeeper of mast cell degranulation

Abstract

To clarify the role that the src homology 2-containing inositol phosphatase (SHIP) plays in mast cell degranulation, the gene for SHIP was disrupted by homologous recombination in embryonic stem cells. Bone-marrowderived mast cells from SHIP؉͞؉, ؉͞؊, and ؊͞؊ F 2 littermates were compared. SHIP؊͞؊ mast cells were found to be far more prone to degranulation, after the crosslinking of IgE preloaded cells, than SHIP؉͞؊ or ؉͞؉ cells. Intriguingly, IgE alone also stimulated massive degranulation in SHIP؊͞؊ but not in ؉͞؉ mast cells. This degranulation with IgE alone, which may be due to low levels of IgE aggregates, correlated with a higher and more sustained intracellular calcium level than that observed with SHIP؉͞؉ cells and was dependent upon the entry of extracellular calcium. Immunoprecipitation studies revealed that the addition of IgE alone to normal mast cells stimulates multiple cascades, which are prevented from progressing to degranulation by SHIP. PI 3-kinase inhibitor studies suggested that IgE-induced activation of PI 3-kinase is upstream of the entry of extracellular calcium and that SHIP restricts this entry by hydrolyzing phosphatidylinositol 3,4,5-trisphosphate. These results show the critical role that SHIP plays in setting the threshold for degranulation and that SHIP directly modulates a ''positive-acting'' receptor.The src homology 2 (SH2)-containing inositol phosphatase (SHIP) is a recently cloned hemopoietic specific signaling intermediate (1-4) that becomes tyrosine-phosphorylated in response both to multiple cytokines and to B and T cell receptor engagement (5). It exists in several molecular mass forms, with the lower 135-, 125-and 110-kDa forms being generated from the 145-kDa full length protein in vivo by the cleavage of its proline rich COOH terminus (6). Although all forms become tyrosine-phosphorylated in response to cytokines, only the 145-and 135-kDa species bind the adaptor protein Shc, and only the 110-kDa form is associated with the cytoskeleton (6). The relative amounts of these different forms change with differentiation (7), and it remains to be determined if they carry out distinct functions within hemopoietic cells. Recently, SHIP has been implicated in the negative signaling pathways that abrogate activation in the cells of the immune system. Specifically, it has been shown to inhibit the activation of immune receptors in both mast cells and B cells by binding to the tyrosine-phosphorylated immunoreceptor tyrosine-based inhibition motif (ITIM) of the inhibitory coreceptor Fc␥RIIB and inhibiting the high affinity IgE receptor-(FcR1) and the B cell receptor-induced influx of calcium, respectively (8, 9). However, its role in modulating the biological responses of activating receptors has not yet been determined. To gain further insight into the role that SHIP plays in regulating the responses elicited by these latter receptors, we generated a SHIP knockout mouse by homologous recombination in embryonic stem cells (10). Although these mice are viable and fertile, they ...

Distribution of the Src-homology-2-domain-containing inositol 5-phosphatase SHIP-2 in both non-haemopoietic and haemopoietic cells and possible involvement of SHIP-2 in negative signalling of B-cells

“…Various models of SHIP-1 mechanism of action have been presented in B-cells [5,7] and mast cells [5,34,35] ; these could therefore be re-evaluated in the light of possible competitive interactions between Shc and SHIP-1 or SHIP-2.…”

R‐form LPS, the master key to the activation ofTLR4/MD‐2‐positive cells

“…MC derived from bone marrow precursor cells of the various mouse strains, were grown in the presence of 1% X63Ag8-653-conditioned medium as a source of IL-3 [72] as described [73]. Differentiated, c-kit and FceR1-positive MC were preloaded overnight with IgE (0.2 lg/mL) and subsequently stimulated in RPMI 1640 (10 6 cells/mL) in duplicates or triplicates with the agents under test.…”

Loss of a single allele of SHIP exacerbates the immunopathology of Pten heterozygous mice

“…These reaction products allow translocation of multiple pleckstrin homology (PH) domain-containing proteins to the membrane, and these in turn regulate downstream events such as proliferation, apoptosis, protein synthesis, and cell shape (reviewed in Chan et al 12 and Martin 13 ). The levels of PIP 3 and PIP 2 are regulated via the actions of Pten, a 3 0 -inositol phosphatase, and SHIP, a 5 0 -inositol phosphatase 14,15 and recent studies have provided evidence of a role for negative regulators of PIP 3 in the maintenance of immune tolerance. While complete deletion of Pten results in embryonic lethality, [16][17][18] Pten +/À mice develop an autoimmune phenotype characterized by lymphadenopathy and splenomegaly that has been attributed to defective B-and T-cell apoptosis.…”