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The Secretion and Uptake of Lysosomal Phospholipase A₂ by Alveolar Macrophages¹

Akira Abe^*, Robert Kelly^*, Jessica Kollmeyer^*, Miki Hiraoka, Ye Lu^* and James A. Shayman^2,*

^* Nephrology Division, Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109 and Department of Opthamology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo

Macrophages have long been known to secrete a Phospholipase A₂ with an acidic pH optimum in response to phagocytic stimuli. However, the enzyme or enzymes responsible for this activity have not been identified. We report that mouse alveolar macrophages release lysosomal phospholipase A₂ (LPLA₂) into the medium of cultured cells following stimulation with zymosan. The release of the enzyme was detected by enzymatic activity assays as well as by Western blotting using an Ab against mouse LPLA₂. LPLA₂ is a high mannose type glycoprotein found in lysosomes, suggesting that the released enzyme might be reincorporated into alveolar macrophages via a mannose or mannose phosphate receptor. Recombinant glycosylated mouse LPLA₂ produced by HEK293 cells was applied to LPLA₂-deficient (LPLA₂^–/–) mouse alveolar macrophages. The uptake of exogenous LPLA₂ into LPLA₂^–/– alveolar macrophages occurred in a concentration-dependent manner. The LPLA₂ taken into the alveolar macrophages colocalized with the lysosomal marker, Lamp-1. This uptake was significantly suppressed in the presence of -methyl-mannoside but not in the presence of mannose 6-phosphate. Thus, the predominant pathway for uptake of exogenous LPLA₂ is via the mannose receptor, with subsequent translocation into acidic, Lamp-1-associated compartments. LPLA₂^–/– alveolar macrophages are characterized by marked accumulation of phosphatidylcholine and phosphatidylethanolamine. Treatment with the recombinant LPLA₂ rescued the LPLA₂^–/– alveolar macrophages by markedly decreasing the phospholipid accumulation. The application of a catalytically inactive LPLA₂ revealed that the enzymatic activity of LPLA₂ was required for the phospholipid reduction. These studies identify LPLA₂ as a high m.w.-secreted Phospholipase A₂.

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 a Merit Review Award from the Department of Veterans Affairs and the Tricorridor Research Fund from the State of Michigan.

² Address correspondence and reprint requests to Dr. James A. Shayman, Department of Internal Medicine, Room 1560 MSRBII, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0676. E-mail address: jshayman{at}umich.edu

³ Abbreviations used in this paper: sPLA2, secreted Phospholipase A₂; LPLA₂, lysosomal Phospholipase A₂; AM, alveolar macrophage; NAS, N-acetylsphingosine; EEA1, early endosomal Ag 1; DAPI, 4',6-diamidino-2-phenylindole.

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				A. Abe and J. A. Shayman The role of negatively charged lipids in lysosomal phospholipase A2 function J. Lipid Res., October 1, 2009; 50(10): 2027 - 2035. [Abstract] [Full Text] [PDF]