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Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles
Published on: June 8, 2012
Cdc42 and RhoB activation are required for mannose receptor-mediated phagocytosis by human alveolar macrophages
Jianmin Zhang1, Jinping Zhu, Xia Bu
1Division of Pulmonary and Critical Care Medicine, Department of Medicine, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA 02115, USA.
Abstract:
Human alveolar macrophages (AMs) phagocytose Pneumocystis (Pc) organisms predominantly through mannose receptors, although the molecular mechanism mediating this opsonin-independent process is not known. In this study, using AMs from healthy individuals, Pc phagocytosis was associated with focal F-actin polymerization and Cdc42, Rac1, and Rho activation in a time-dependent manner. Phagocytosis was primarily dependent on Cdc42 and RhoB activation (as determined by AM transfection with Cdc42 and RhoB dominant-negative alleles) and mediated predominantly through mannose receptors (as determined by siRNA gene silencing of AM mannose receptors). Pc also promoted PAK-1 phosphorylation, which was also dependent on RhoGTPase activation. HIV infection of AMs (as a model for reduced mannose receptor expression and function) was associated with impaired F-actin polymerization, reduced Cdc42 and Rho activation, and markedly reduced PAK-1 phosphorylation in response to Pc organisms. In healthy AMs, Pc phagocytosis was partially dependent on PAK activation, but dependent on the Rho effector molecule ROCK. These data provide a molecular mechanism for AM mannose receptor-mediated phagocytosis of unopsonized Pc organisms that appears distinct from opsonin-dependent phagocytic receptors. Reduced AM mannose receptor-mediated Cdc42 and Rho activation in the context of HIV infection may represent a mechanism that contributes to the pathogenesis of opportunistic pneumonia.
Insights
Human alveolar macrophages use mannose receptors to engulf Pneumocystis organisms. This process involves actin polymerization and specific protein activation, which is impaired in HIV infection, potentially leading to pneumonia.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- Human alveolar macrophages (AMs) phagocytose Pneumocystis (Pc) organisms via mannose receptors.
- The molecular mechanisms of this opsonin-independent phagocytosis are not fully understood.
Purpose of the Study:
- To elucidate the molecular mechanism of mannose receptor-mediated Pneumocystis phagocytosis by human alveolar macrophages.
- To investigate the role of Rho GTPases and actin polymerization in this process.
- To examine the impact of HIV infection on Pneumocystis phagocytosis by AMs.
Main Methods:
- AMs from healthy individuals and HIV-infected individuals were used.
- Techniques included F-actin polymerization assays, Rho GTPase activation assays (Cdc42, Rac1, Rho), siRNA gene silencing of mannose receptors, dominant-negative allele transfection, and Western blotting for PAK-1 phosphorylation.
- Phagocytosis assays were performed to assess Pc uptake.
Main Results:
- Pc phagocytosis in healthy AMs involved focal F-actin polymerization and activation of Cdc42, Rac1, and Rho.
- Phagocytosis was dependent on Cdc42 and RhoB activation and mediated by mannose receptors.
- Pc promoted PAK-1 phosphorylation, dependent on Rho GTPase activation.
- HIV infection impaired F-actin polymerization, Cdc42/Rho activation, and PAK-1 phosphorylation in response to Pc.
- Pc phagocytosis in healthy AMs was partially dependent on PAK activation and dependent on the Rho effector ROCK.
Conclusions:
- A molecular mechanism for mannose receptor-mediated phagocytosis of unopsonized Pc by AMs was identified, distinct from opsonin-dependent pathways.
- Reduced mannose receptor-mediated Cdc42 and Rho activation in HIV-infected AMs may contribute to Pneumocystis pneumonia pathogenesis.
