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Measurement of active phagocytosis by polymorphonuclear leukocytes by fluorescence liberation from phagocytized
Abstract:
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.
Insights
A new assay quantifies phagocytosis by polymorphonuclear leukocytes (PMN) using fluorescent microspheres. This method measures beta-glucuronidase activity within phagosomes, offering a sensitive tool for studying immune cell function.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Phagocytosis is a critical cellular process for immune defense.
- Quantifying phagocytosis by polymorphonuclear leukocytes (PMN) requires sensitive and accurate methods.
- Existing methods may not fully capture the dynamics of phagosome-lysosome interactions.
Purpose of the Study:
- To develop and validate a novel fluorescence-based assay for measuring phagocytosis by PMN.
- To characterize the optimal conditions for this new assay using specific microspheres.
- To demonstrate the utility of the assay in quantifying beta-glucuronidase activity within phagosomes.
Main Methods:
- Conjugation of 4-methylumbelliferyl-beta-D-glucuronide (4MUGL) to 2.0-micron glyceryl-methacrylate microspheres.
- Measurement of liberated 4-methylumbelliferone (4MU) fluorescence following phagocytosis by PMN.
- Comparison of six types of 4MUGL-microspheres for their liberating activity.
- Determination of optimal incubation time, microsphere concentration, and enzyme activity.
Main Results:
- Four types of 4MUGL-microspheres showed significant phagocytic activity and liberated 4MU.
- 4MUGL-MS610 exhibited the highest liberating activity and was used for further optimization.
- Liberation of 4MU increased linearly with incubation time (0-60 min) and microsphere concentration (up to 4 X 10(8)/ml).
- The assay demonstrated sensitivity to approximately 50 pmol/ml, correlating with phagocytized microspheres.
Conclusions:
- The developed fluorescence assay accurately measures phagocytosis by PMN.
- 4MU liberation is indicative of beta-glucuronidase activity within PMN phagosomes.
- This assay provides a sensitive and quantitative method for studying phagocytic processes and lysosomal enzyme activity.