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Updated: Jul 3, 2026
Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages
Published on: May 24, 2019
Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: analysis by scanning cytometry
Timothy H Sulahian1, Amy Imrich, Glen Deloid
1Harvard School of Public Health, Molecular and Integrative Physiological Sciences Program, 655 Huntington Ave, Building II, 2nd Floor, Boston, MA 02115, USA. tsulahian@cellsignal.com
Background:
Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Mice with genetic deletions of scavenger receptors, such as SR-A and MARCO, are susceptible to infection or inflammation from inhaled pathogens or dusts. However, the signaling pathways required for scavenger receptor-mediated phagocytosis of unopsonized particles have not been characterized.
Methods:
We developed a scanning cytometry-based high-throughput assay of macrophage phagocytosis that quantitates bound and internalized unopsonized latex beads. This assay allowed the testing of a panel of signaling inhibitors which have previously been shown to target opsonin-dependent phagocytosis for their effect on unopsonized bead uptake by human in vitro-derived alveolar macrophage-like cells. The non-selective scavenger receptor inhibitor poly(I) and the actin destabilizer cytochalasin D were used to validate the assay and caused near complete abrogation of bead binding and internalization, respectively.
Results:
Microtubule destabilization using nocodazole dramatically inhibited bead internalization. Internalization was also significantly reduced by inhibitors of tyrosine kinases (genistein and herbimycin A), protein kinase C (staurosporine, chelerythrine chloride and Gö 6976), phosphoinositide-3 kinase (LY294002 and wortmannin), and the JNK and ERK pathways. In contrast, inhibition of phospholipase C by U-73122 had no effect.
Conclusion:
These data indicate the utility of scanning cytometry for the analysis of phagocytosis and that phagocytosis of unopsonized particles has both shared and distinct features when compared to opsonin-mediated phagocytosis.
Insights
Scavenger receptors on lung macrophages engulf unopsonized particles. Signaling pathways involving microtubules, tyrosine kinases, protein kinase C, PI3K, JNK, and ERK are crucial for this innate immune process.
Area of Science:
- Immunology
- Cell Biology
Background:
- Scavenger receptors (SRs) are key innate immune components in the lung.
- Alveolar macrophages utilize SRs to bind and phagocytose unopsonized targets.
- Genetic deficiencies in SRs (e.g., SR-A, MARCO) increase susceptibility to inhaled pathogens and dusts.
Purpose of the Study:
- To characterize signaling pathways involved in scavenger receptor-mediated phagocytosis of unopsonized particles.
- To establish and validate a high-throughput assay for analyzing macrophage phagocytosis.
Main Methods:
- Developed a scanning cytometry assay to quantify phagocytosis of unopsonized latex beads by macrophage-like cells.
- Utilized signaling inhibitors to assess their impact on unopsonized particle uptake.
- Validated the assay using poly(I) (scavenger receptor inhibitor) and cytochalasin D (actin destabilizer).
Main Results:
- Microtubule destabilization significantly inhibited particle internalization.
- Inhibitors of tyrosine kinases, protein kinase C, phosphoinositide-3 kinase (PI3K), JNK, and ERK pathways markedly reduced bead internalization.
- Inhibition of phospholipase C did not affect particle uptake.
Conclusions:
- Scanning cytometry is a valuable tool for phagocytosis analysis.
- Phagocytosis of unopsonized particles shares some signaling pathways with opsonin-mediated phagocytosis but also has distinct features.
