Measurement of active phagocytosis by polymorphonuclear leukocytes by fluorescence liberation from phagocytized microspheres.
Phagocytosis by polymorphonuclear leukocytes (PMN) was determined by a newly developed technique based on measurement of liberation of a fluorescence substance from PMN phagosomes; 4-methylumbelliferyl-beta-D-glucuronide (4MUGL), which is a substrate of beta-glucuronidase in lysosome, was conjugated with a microsphere, and 4-methylumbelliferone (4MU) liberated from phagocytized 4MUGL-microspheres was measured. The microspheres were composed of glyceryl-methacrylate having a diameter of 2.0 micron. Liberating activity of six kinds of 4MUGL-microspheres containing various amounts of amino and carboxyl groups was compared. Among these six kinds of 4MUGL-microspheres, four kinds showed activity similar to that of morphological phagocytosis. These four kinds of 4MUGL-microspheres liberated 4MU into the extracellular fluid from PMN during phagocytosis. Furthermore, they were recognized as a substrate of purified beta-glucuronidase. 4MUGL-MS610 showed the highest liberating activity among the four kinds of microspheres. Optimal conditions for phagocytosis by PMN were determined using 4MUGL-MS610. Total liberation of 4MU from the microspheres increased almost linearly with incubation time with PMN from 0 to 60 min and was linear with 4MUGL-MS in concentrations up to 4 X 10(8) microspheres/ml. This liberation was parallel to phagocytosis in a dose-dependent fashion. During 10-min incubation 20.4% of 4MU was liberated from 4MUGL-microspheres with phagocytosis. Seventy-five percent of the liberated 4MU was distributed in the extracellular fluid. 4MU distributed in the extracellular fluid was not attributable to hydrolysis of unphagocytized microspheres by beta-glucuronidase extracellularly leaked from PMN by phagocytosis. Also phagocytized 4MUGL-MS610 by PMN was observed by scanning electron microscopy. These results indicate that 4MU was liberated from 4MUGL-MS by hydrolysis due to beta-glucuronidase released into phagosomes with phagocytosis by PMN. Sensitivity of this assay was limited to about 50 pmol/ml, being less than 0.5-1 microsphere phagocytized into one cell.
D002463:Cell Membrane Permeability; D005110:Extracellular Space / Q000032:analysis; D005966:Glucuronidase / Q000378:metabolism; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008863:Microspheres; D009504:Neutrophils / Q000502:physiology*; D009195:Peroxidase / Q000378:metabolism; D010587:Phagocytosis; D013050:Spectrometry, Fluorescence / Q000379:methods; D014468:Umbelliferones
D014468:Umbelliferones; D006923:Hymecromone; D009195:Peroxidase; D005966:Glucuronidase