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Updated: Apr 27, 2026
A Quantitative Assessment of the Phagocytosis of Allogeneic and Xenogeneic Erythrocytes by Rat Macrophages In Vitro
Published on: August 22, 2025
Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in
Karrie Tartaro1, Maria VanVolkenburg, Dean Wilkie
1Pfizer Worldwide Research and Development, Drug Safety Research and Development , Groton, CT 06340 , USA.
Abstract:
The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles(®)). Fluorescence of the Bioparticles(®) is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles(®) phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS(®) Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles(®). The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles(®). In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the detection of inhibition of splenic macrophage function will require further assay development.
Insights
A new non-radioactive method effectively measures mononuclear phagocyte system (MPS) function in rats by tracking fluorescent E. coli particles in the liver. This assay can assess how compounds impact immune cell activity, though spleen function requires further development.
Area of Science:
- Immunology
- Pharmacology
- Toxicology
Background:
- The mononuclear phagocyte system (MPS) is crucial for host defense against pathogens.
- Inhibition of MPS function can impair the clearance of microbes and increase infection susceptibility.
- Current methods for assessing MPS function are often radioactive or labor-intensive.
Purpose of the Study:
- To develop a novel, non-radioactive, quantitative in vivo method for measuring MPS function.
- To utilize pH-sensitive fluorescent E. coli bioparticles for phagocytosis assessment.
- To evaluate the sensitivity of the developed method using known MPS-suppressing agents.
Main Methods:
- Heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles®) were administered to rats.
- Phagocytosis was quantified by measuring fluorescence intensity in liver and spleen ex vivo using IVIS imaging.
- Peripheral blood neutrophil phagocytosis was assessed via flow cytometry.
- Rats were pre-treated with MPS-suppressing compounds (clodronate liposomes, latex particles, MVE polymer) before Bioparticle® administration.
Main Results:
- Bioparticles® co-localized with macrophage markers in the liver but not the spleen.
- All tested compounds reduced phagocytosis in the liver.
- Splenic phagocytic activity showed no consistent inhibition across tested compounds.
- Clodronate liposomes and MVE polymer increased neutrophil phagocytosis in peripheral blood.
Conclusions:
- A new in vivo rat model effectively measures hepatic MPS phagocytosis using fluorescent E. coli bioparticles.
- The model can assess the impact of various compounds on liver-based MPS function.
- Further assay development is needed to reliably detect inhibition of splenic macrophage function.
