Abstract
The aim of this study was to evaluate the effects of chloroquine on phagolysosomal fusion (PLF) in cultured guinea pig alveolar macrophages (AMs). This technique may be of significance for antitubercular drugs, because the survival of Mycobacterium tuberculosis is linked to evasion of PLF. Guinea pig AMs were obtained from anesthetized animals after exsanguination. The AMs were cultured at a density of 1×106 cell/mL in 24-well plates after attachment to 13-mm coverslips. Culture conditions were at 37°C, with 95% air/5% CO2 in Roswell Park Memorial Institute (RPMI) 1640 medium with 10% heat-inactivated fetal bovine serum. Rhodamine-dextran (70 kd) was incubated with the cells at 0.25 mg/mL for 24 hours to label the lysosomes. Chloroquine treatment where indicated was performed at 10–20 μ g/mL for 1 hour. Fluorescent BioParticles were then added, and PLF was monitored by formation of an organge-yellow fluorescence on fusion of green fluorescent BioParticles with rhodamine-labeled lysosomes. PLF endpoints were measured by scoring for the percentage of orange-yellow cells in the field of view. Image analysis to measure the intensity of the orange-yellow color was performed by obtaining a, b values for 5×5 pixel areas using the Photo Adobe program 4.0.1.
The results indicated that the rate of PLF was enhanced by chloroquine. Thus, chloroquine may be used to potentiate the effects of rifampicin. This may be confirmed by studies involving similar dual fluorophore labeling techniques of fluorescein-labeled formulation in macrophages infected with M. tuberculosis. Preliminary studies with the rhodamine-labeled formulation confirmed cellular uptake and persistence for up to 7 days in culture.
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References
Kochi A. The global tuberculosis situation and the new control strategy of the World Health Organization. Tubercle. 1991;72:1–6.
Bloom BR, Murray CJL. Tuberculosis: commentary on a resurgent killer. Science. 1992;257:1055–1064.
Dannenberg AM Jr, Tomashefski JF Jr. Pathogenesis of pulmonary tuberculosis. In: Fishman AP, ed. Pulmonary Diseases and Disorders. Vol 3. New York: McGraw-Hill, 1988:821–842.
Dannenberg AM Jr, Rook GAW. Pathogenesis of pulmonary tuberculosis: an interplay of tissue-damaging and macrophage activating immune responses and dual mechanisms that control bacillary multiplication. In: Bloom BR, ed. Tuberculosis: Pathogenesis. Protection and Control Washington DC: American Society for Microbiology. 1994:459–483.
D'Arcy Hart P, Young MR. Manipulations of phagosome-lysosome fusion in cultured macrophages: potentialities and limitations. In: van Furth R, ed. Mononuclear Phagocytes: Functional Aspects. The Hague: Martinus Nijhoff, 1980:1039–1055.
Brubaker RR. Mechanisms of bacterial virulence. Ann Rev Microbiol. 1985;39:21–50.
Goren MB, D'Arcy Hart P, Young MR, Armstrong JA. Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides ofMycobacterium tuberculosis. Proc Natl Acad Sci USA 1976:78:2510–2514.
Gordon AH, D'Arcy Hart P, Young MR. Ammonia inhibits phagosome-lysosome fusion in macrophages. Nature. 1980;280:79–80.
D'Arcy Hart P, Young MR, Gordon AH, Sullivan KH. Inhibition of phagosome-lysome fusion in macrophages by certain Mycobacteria can be explained by inhibition of lysosomal movements observed after phagocytosis. J Exp Med. 1987;166:933–946.
D'Arcy Hart P, Young MR. Mampulations of the phagosome-lysome fusion response in cultured macrophages Enhancement of fusion by chloroquine and other amines. Exp Cell Res. 1978;114:486–490.
Duzgunes N, Majumdar S, Goren MB. Fluorescence methods for monitoring phagosome-lysosome fusion in human macrophages. Meth Enzymol. 1993;221:234–238.
O'Donnell PB, McGinity JW. Preparation of microspheres by the solvent evaporation technique. Advanced Drug Delivery Reviews 1997;28:25–42.
Hausberger AG, Kenley RA, DeLuca PP. Gamma irradiation effects on molecular weights and in vitro degradation of poly (D,L-lactide-coglycolide) microparticles. Pharm Res. 1995;12:851–856.
Hickey AJ. Methods of aerosol particle size characterization. In: Hickey AJ, ed. Pharmaceutical Inhalation Aerosol Technology. New York: Marcel Dekker, 1992:219–253.
Denkbas EB, Kaitian X, Tuncel A, Piskin E. Rifampicin-carrying poly (D,L-lactide) microspheres: loading and release. J Biomater Sci Polymer Edn. 1994;6:815–825.
Tabata Y, Ikada Y. Macrophage phagocytosis of biodegradable microspheres composed of L-lactic acid/glycolic acid homo- and copolymers. J Biomedical Materials Res. 1988;22:837–858.
Kielian MC, Cohn ZA. Determinants of phagosome-lysosome fusion in mouse macrophages in cultured macrophages: potentialities and limitations. In: van Furth R, ed. Mononuclear Phagocytes: Functional Aspects. The Hague: Martinus Nijhoff, 1980:1077–1102.
Adobe Photoshop 3.0 User Guide Mountainview, CA: Adobe Systems Inc.; 1994:7–11.
Bruce MC, Honaker CE, Cross RJ. Lung fibroblasts undergo apoptosis following alveolarization. Am J Respir Cell Mol Biol. 1999;20:228–236
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Published: Novemeer 14, 2000.
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Bhat, M., Hickey, A.J. Effect of chloroquine on phagolysosomal fusion in cultured guinea pig alveolar macrophages: Implications in drug delivery. AAPS PharmSci 2, 34 (2000). https://doi.org/10.1208/ps020434
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DOI: https://doi.org/10.1208/ps020434