title: Signaling pathways required for macrophage scavenger receptor-mediated phagocytosis: analysis by scanning cytometry.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Harvard School of Public Health, Molecular and Integrative Physiological Sciences Program, 655 Huntington Ave, Building II, 2nd Floor, Boston, MA 02115, USA. tsulahian@cellsignal.com
authors.forename: Timothy H
authors.initials: TH
authors.lastname: Sulahian
authors.forename: Amy
authors.initials: A
authors.lastname: Imrich
authors.forename: Glen
authors.initials: G
authors.lastname: Deloid
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Winkler
authors.forename: Lester
authors.initials: L
authors.lastname: Kobzik
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D006801:Humans; D048670:MAP Kinase Kinase 4 / Q000037:antagonists & inhibitors / Q000502:physiology; D020930:MAP Kinase Kinase Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D016676:Macrophages, Alveolar / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008863:Microspheres; D008870:Microtubules / Q000187:drug effects / Q000502:physiology; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D015739:Nocodazole / Q000494:pharmacology; D010587:Phagocytosis / Q000187:drug effects / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011069:Poly I / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011494:Protein Kinases / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology; D051116:Receptors, Scavenger / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D050257:Tubulin Modulators / Q000494:pharmacology
chemical_list: D015638:Cytochalasin D; D048670:MAP Kinase Kinase 4; D020930:MAP Kinase Kinase Kinases; D015739:Nocodazole; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011069:Poly I; D011493:Protein Kinase C; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D051116:Receptors, Scavenger; D050257:Tubulin Modulators