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SHIP-1 Increases Early Oxidative Burst and Regulates Phagosome Maturation in Macrophages¹

Lynn A. Kamen^*,, Jonathan Levinsohn^*, Amy Cadwallader^*, Susheela Tridandapani and Joel A. Swanson^2,*,

^* Department of Microbiology and Immunology and Program in Immunology, University of Michigan Medical School, Ann Arbor, MI 48109; and Department of Internal Medicine, Ohio State University, Columbus, OH 43210

Although the inositol phosphatase SHIP-1 is generally thought to inhibit signaling for Fc receptor-mediated phagocytosis, the product of its activity, phosphatidylinositol 3,4 bisphosphate (PI(3,4)P₂), has been implicated in activation of the NADPH oxidase. This suggests that SHIP-1 positively regulates the generation of reactive oxygen species after phagocytosis. To examine how SHIP-1 activity contributes to Fc receptor-mediated phagocytosis, we measured and compared phospholipid dynamics, membrane trafficking, and the oxidative burst in macrophages from SHIP-1-deficient and wild-type mice. SHIP-1-deficient macrophages showed significantly elevated ratios of PI(3,4,5)P₃ to PI(3,4)P₂ on phagosomal membranes. Imaging reactive oxygen intermediate activities in phagosomes revealed decreased early NADPH oxidase activity in SHIP-1-deficient macrophages. SHIP-1 deficiency also altered later stages of phagosome maturation, as indicated by the persistent elevation of PI(3)P and the early localization of Rab5a to phagosomes. These direct measurements of individual organelles indicate that phagosomal SHIP-1 enhances the early oxidative burst through localized alteration of the membrane 3'-phosphoinositide composition.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by National Institutes of Health Grants AI35950 and AI64668 (to J.S.) and NCI P01 CA095426 (to S.T.).

² Address correspondence and reprint requests to Dr. Joel A. Swanson, University of Michigan Medical School, Medical Sciences II Building, Room 5818, Ann Arbor, MI 48109. E-mail address: jswan{at}umich.edu

³ Abbreviations used in this paper: 3'-PI, 3'-phosphoinositide; BMDM, bone marrow-derived macrophage; Btk, Bruton's tyrosine kinase; CIT, citrine; CFP, cyan fluorescent protein; FRET, fluorescence resonance energy transfer; Lamp-1, lysozyme-associated membrane protein-1; PH, pleckstrin homology; PI(3)P, phosphatidylinositol 3-phosphate; PI(3,4)P₂, phosphatidylinositol 3,4 bisphosphate; PI(3,4,5)P₃, phosphatidylinositol 3,4,5-trisphosphate; PLC, phospholipase C; R_C, ratio image in the cell; RFP, red fluorescent protein; R_M, ratio image; ROI, reactive oxygen intermediate; R_M, ratio image in phagosome; Tapp, tandem PH domain-containing protein; YFP, yellow fluorescent protein; R_P, ratio image in phagosome.

⁴ The online version of this article contains supplemental material.