The recessive mutation 'Heedless' (hdl) was detected in third-generation N-ethyl-N-nitrosourea-mutated mice that showed defective responses to microbial inducers. Macrophages from Heedless homozygotes signaled by the MyD88-dependent pathway in response to rough lipopolysaccharide (LPS) and lipid A, but not in response to smooth LPS. In addition, the Heedless mutation prevented TRAM-TRIF-dependent signaling in response to all LPS chemotypes. Heedless also abolished macrophage responses to vesicular stomatitis virus and substantially inhibited responses to specific ligands for the Toll-like receptor 2 (TLR2)-TLR6 heterodimer. The Heedless phenotype was positionally ascribed to a premature stop codon in Cd14. Our data suggest that the TLR4-MD-2 complex distinguishes LPS chemotypes, but CD14 nullifies this distinction. Thus, the TLR4-MD-2 complex receptor can function in two separate modes: one in which full signaling occurs and one limited to MyD88-dependent signaling.

Although IgE binding to mast cells is thought to be a passive presensitization step, we demonstrate herein that monomeric IgE (mIgE) in the absence of antigen (Ag) stimulates multiple phosphorylation events in normal murine bone marrow-derived mast cells (BMMCs). While mIgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE + Ag. Moreover, mIgE prevents the apoptosis of cytokine-deprived BMMCs, likely by maintaining Bcl-X(L) levels and producing autocrine-acting cytokines. The addition of Ag does not increase this IgE-induced survival. Since IgE concentrations as low as 0.1 microg/ml enhance BMMC survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals.

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 ...

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