The Secretion and Uptake of Lysosomal Phospholipase A2 by Alveolar Macrophages

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 α-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.