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The role of SHIP in macrophage differentiation and function Rauh, Michael J.

Abstract

The SH2 containing inositol 5'-phosphatase (SHIP) is a hemopoieticspecific protein that catalyzes the hydrolysis of the phosphatidylinositol 3-kinase (PI3K)-generated second messenger PI-3,4,5-P₃ (PIP₃), to PI-3,4-P₂ (PIP₂) and thereby negatively regulates hemopoietic cell survival, proliferation, differentiation and activation. Herein, macrophage development and function were compared in SHIP+/+ and -/- mice. SHIP was found to restrain in vitro bone marrowderived macrophage (BMMφ) survival (or proliferation) and differentiation, consistent with the increased number of macrophages observed in SHIP-/- mice. We also compared the function of J2 virus-transformed SHIP+/+ and -/- BMMφ cell lines and found that SHIP-/- J2M BMMφ cell lines (-/-J2Ms) were functionally impaired in inducible nitric oxide (NO) synthase (iNOS) induction and high-output NO production, an important, classical (M1) macrophage activation strategy to combat the growth of tumours and microorganisms. This was ascribed to deficient nuclear localization of IRF1 and inhibition of iNOS transcription in these transformed macrophages. In contrast, primary SHIP-/- BMMφs routinely demonstrated enhanced LPS-stimulated iNOS/NO induction, likely as a result of PI3K-mediated enhancement of the p70S6K/IFNβ/Stat1/iNOS pathway. Differential impacts upon this axis also provided an explanation for the opposite effects of the PI3K inhibitors, LY294002 and wortmannin, on iNOS/NO. We also found that SHIP-/- BMMφs failed to tolerize to a second dose of LPS, likely because SHIP protein levels were upregulated in wild-type BMMφs in an autocrine-acting, TGF(3-mediated tolerance loop. Analysis of in vivo-differentiated, resident peritoneal and alveolar macrophages (PMφs, AMφs) from SHIP-/- mice revealed impaired NO generation, despite sufficient iNOS induction, due to constitutive arginase Imediated L-arginine substrate competition, which redirected L-arginine metabolism away from cytotoxic NO and towards the production of healing/inflammation-resolving intermediates. These and other features were recognized as alternative (M2) macrophage activation. Consistent with pathological M2-skewing in SHIP-/- mice, their lungs were fibrotic and contained macrophage-associated Ym1 crystals. Moreover, implanted tumours grew more rapidly in the M2-skewed environment of SHIP-/- mice. Interestingly, BMMφs from SHIP-/- mice did not display this M2 phenotype unless exposed to TGF(3- containing mouse plasma early during in vitro differentiation, suggesting that an environment of elevated PIP₃ and TGFβ arising during in vivo macrophage differentiation may contribute to M2-skewing.

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