Abstract
BackgroundScavenger 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.MethodsWe 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.ResultsMicrotubule 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.ConclusionThese 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.
Highlights
Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets
Characterization of GM-CSF matured MØ (GM-MØ) Monocytes are typically matured into MØ in vitro using MCSF
We followed the GM-CSF-based differentiation protocol of Akagawa, et al, designed to produce monocyte-derived MØ with a distinctly Alveolar macrophages (AMs)-like phenotype (GM-MØ) [48]. Both AM and GM-MØ have been shown to produce lower levels of H2O2, express higher levels of catalase and are more resistant to H2O2 toxicity when compared to M-CSF derived MØ
Summary
Scavenger receptors are important components of the innate immune system in the lung, allowing alveolar macrophages to bind and phagocytose numerous unopsonized targets. Scavenger receptors (SRs) are a key component of the innate immune system In addition to their well-known role in low-density lipoprotein metabolism, SRs play a critical role in AM clearance of inhaled particles by binding and allowing the cells to internalize unopsonized microorganisms, apoptotic bodies and environmental dusts [6,7]. It is important to point out that AMs express several other less well-characterized SRs including LOX-1, SR-PSOX and SRCL [10] These SRs are capable of binding bacteria [26,27,28] and might contribute to the AM response to inhaled insults
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