ABSTRACT
Aspergillus fumigatus is an opportunistic fungal pathogen causing detrimental infections in immunocompromised individuals. Dendritic cells (DCs) are potent antigen-presenting cells and recognize the A. fumigatus cell wall component ß-1,3 glucan via Dectin-1, followed by DC maturation and cytokine release. Here, we demonstrate that human primary myeloid DCs (mDCs) interact with different morphotypes of A. fumigatus. Dectin-1 is expressed on mDCs and is down-regulated after contact with A. fumigatus, indicating that mDCs recognize A. fumigatus via this receptor. Blocking of Dectin-1, followed by stimulation with depleted zymosan diminished the up-regulation of the T-cell co-stimulatory molecules CD40, CD80, HLA-DR and CCR7 on mDCs and led to decreased release of the cytokines TNF-α, IL-8, IL-1ß and IL-10.
Subject(s)
Aspergillus fumigatus/immunology , Dendritic Cells/immunology , Down-Regulation/immunology , Host-Pathogen Interactions/immunology , Lectins, C-Type/immunology , Cells, Cultured , Cytokines/biosynthesis , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Dendritic Cells/microbiology , Flow Cytometry , Humans , Lectins, C-Type/antagonists & inhibitors , Lectins, C-Type/drug effects , Lectins, C-Type/metabolism , Zymosan/analogs & derivatives , Zymosan/pharmacologyABSTRACT
Mutations at the natural resistance-associated macrophage protein 1 (Nramp1) locus cause susceptibility to infection with antigenically unrelated intracellular pathogens. Nramp1 codes for an integral membrane protein expressed in the lysosomal compartment of macrophages, and is recruited to the membrane of phagosomes soon after the completion of phagocytosis. To define whether Nramp1 functions as a transporter at the phagosomal membrane, a divalent cation-sensitive fluorescent probe was designed and covalently attached to a porous particle. The resulting conjugate, zymosan-FF6, was ingested by macrophages and its fluorescence emission was recorded in situ after phagocytosis, using digital imaging. Quenching of the probe by Mn(2+) was used to monitor the flux of divalent cations across the phagosomal membrane in peritoneal macrophages obtained from Nramp1-expressing (+/+) and Nramp1-deficient (-/-) macrophages. Phagosomes from Nramp1(+/+) mice extrude Mn(2+) faster than their Nramp(-/-) counterparts. The difference in the rate of transport is eliminated when acidification of the phagosomal lumen is dissipated, suggesting that divalent metal transport through Nramp1 is H(+) dependent. These studies suggest that Nramp1 contributes to defense against infection by extrusion of divalent cations from the phagosomal space. Such cations are likely essential for microbial function and their removal from the phagosomal microenvironment impairs pathogenesis, resulting in enhanced bacteriostasis or bactericidal activity.
Subject(s)
Carrier Proteins/metabolism , Cation Transport Proteins , Intracellular Membranes/metabolism , Iron-Binding Proteins , Macrophages, Peritoneal/metabolism , Manganese/metabolism , Membrane Proteins/metabolism , Phagosomes/immunology , Phagosomes/metabolism , Animals , Biological Transport/drug effects , Cations, Divalent/metabolism , Ethylenediamines/pharmacology , Fluorescent Dyes/chemical synthesis , Fluorescent Dyes/metabolism , Fura-2/metabolism , Hydrogen-Ion Concentration , Intracellular Membranes/drug effects , Macrophages, Peritoneal/cytology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/immunology , Mice , Mice, Knockout , Microscopy, Fluorescence , Mutation , Spectrometry, Fluorescence , Thapsigargin/pharmacology , Zymosan/analogs & derivatives , Zymosan/chemical synthesis , Zymosan/metabolismABSTRACT
At the surface of phagocytes, antibody-opsonized particles are recognized by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocytosis which is poorly defined. We have analyzed the function of the Rho proteins Rac1 and CDC42 in the high affinity receptor for IgE (FcepsilonRI)-mediated phagocytosis using transfected rat basophil leukemia (RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rac1. Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the particles and further, to particle internalization. This process was inhibited by Clostridium difficile toxin B, a general inhibitor of Rho GTP-binding proteins. Dominant inhibition of Rac1 or CDC42 function severely inhibited particle internalization but not F-actin accumulation. Inhibition of CDC42 function resulted in the appearance of pedestal-like structures with particles at their tips, while particles bound at the surface of the Rac1 mutant cell line were enclosed within thin membrane protrusions that did not fuse. These phenotypic differences indicate that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup. Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phosphorylated proteins around bound particles. Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine dephosphorylation was inhibited by treatment of RBL-2H3 cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases. Altogether, our data show that Rac1 and CDC42 are required to coordinate actin filament organization and membrane extension to form phagocytic cups and to allow particle internalization during FcR-mediated phagocytosis. Our data also suggest that Rac1 and CDC42 are involved in phosphotyrosine dephosphorylation required for particle internalization.
