NOVEL DELIVERY OF ANTIOXIDANT ENZYME CATALASE TO ALVEOLAR MACROPHAGESBY FC RECEPTOR-MEDIATED ENDOCYTOSIS

Excessive production of reactive oxygen species by alveolar macrophage s (AMs) in response to inhaled toxic substances is a major cause of ox idative lung injury. Therapeutic approaches designed to protect the lu ngs from oxidative injury by administering native antioxidant enzymes such as catalase and superoxide dismutase have been suggested. However , problems associated with poor penetration of these enzymes to the in tracellular target sites have limited their effective use. The present study reports a drug targeting method based on receptor-mediated endo cytosis of the antioxidant enzyme catalase to the AMs. This method emp loys molecular conjugate consisting of a cognate moiety, in this case IgG which recognizes the macrophage Fc receptor, covalently linked to the enzyme catalase via the reversible disulfide linkage. The uptake e fficiency of the enzyme conjugate and its protection against oxidative injury were evaluated microfluorometrically using the intracellular o xidative probe dichlorodihydrofluorescein BSA: anti BSA antibody compl ex (DCHF-IC), and the cell viability indicator propidium iodide. The D CHF-IC-stimulated macrophages exhibited a dose- and time-dependent inc rease in intracellular fluorescence with a half maximal response dose of approximately 120 mu g/ml. Free catalase (50-500 U/ml) failed to in hibit the DCHF-IC-induced oxidative burst and had only a marginal prot ective effect on AM injury. In contrast, the catalase-IgG conjugate (5 0-500 U/ml) strongly inhibited both the DCHF-IC-induced oxidation and injury in a dose-dependent manner. Effective inhibition was shown to r equire both the antioxidant catalase moiety and the cognate moiety for the cell surface receptor. Specific internalization of the conjugate through the Fc receptor was also investigated by competitive inhibitio n using free IgG. Under this condition, the conjugate showed a much re duced protective effect on intracellular oxidation, indicating conjuga te internalization through the Fc endocytosis pathway. Thus, the enzym e-IgG conjugate system may be used as an effective and selective means to deliver antioxidant enzymes to the intracellular oxidative targets of the AMs.