The macrophage marches on its phagosome: dynamic assays of phagosome function - PubMed (original) (raw)

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The macrophage marches on its phagosome: dynamic assays of phagosome function

David G Russell et al. Nat Rev Immunol. 2009 Aug.

Abstract

Professional phagocytes ingest particulate material to fulfil a diverse array of functions in a multicellular organism. The ancestral function of phagosomes is digestion; however, through evolution this degradative capacity has become pivotal to the adaptive immune response by processing antigens to be presented to lymphocytes. Moreover, phagocytes have also acquired an active role in microbial killing. This Innovation article describes new assays that probe the biological activities which occur within phagosomes. These assays provide functional insights into how the phagosome fulfils its diverse roles in homeostasis and in innate and adaptive immune responses.

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Figures

Figure 1

Figure 1. During maturation, the phagosome forms transient interactions with a wide range of intracellular organelles

Phagosome maturation refers to the process of phagosome remodeling through a series of independent events, following its formation at the surface of the phagocyte, and culminating in the complete fusion of the phagosome with the lysosome. Following engagement of phagocytic receptors, an area of the surface is remodeled around the particle, forming the phagocytic cup. Some protein complexes such as the NADPH oxidase complex, can be recruited and activated prior to phagosomal closure, facilitating a rapid antimicrobial response at the cell surface. The phagosome, once closed, becomes increasingly more acidic, through the accumulation of V ATPases that pump protons into the compartment, and hydrolytically-competent, through the acquisition of lysosomal enzymes. This process is marked by transient fusion events with multiple intracellular organelles including the recycling endosomal machinery, the synthetic–secretory apparatus including the endoplasmic reticulum, secretory lysosomes, and multi-vesicular bodies, which may include the MIIC compartment and/or the autophagosome. Finally, the phagosome fuses with pre-existing, dense lysosomal bodies and equilibrates to a pH of 4.5–5.0.

Figure 2

Figure 2. Real-time measurement of changing phagosomal hydrolase activity in the phagosome during the maturation process

Assays have been developed that measure bulk proteinase activity, cysteine proteinase activity, lipolyis and β-galactosidase activity. The assays involve fluorogenic substrates that are linked to silica beads (A). These beads are also coupled with an opsonizing molecule, such as IgG or mannosylated BSA to facilitate their uptake by macrophages and a calibration fluorochrome. Results are usually expressed as a ratio of substrate fluorescence: calibration fluorescence. The examples illustrated show experiments with beads coupled with the proteinase substrate DQ Green BSA, which consists of albumin derivatized with a self-quenching fluorochrome. The ensuing change in fluorescence generated through the release of fluorescent peptides can be measured by several different instrument, each of which provides its own unique insight. A spectrofluorometer (B) provides a kinetic readout that represents an average value across a population of cells mounted on glass coverslips in cuvettes. A confocal microscope (C) allows visualization and quantitation of the increase in green fluorescence from the hydrolyzed substrate compared with the red fluorescence of the calibration fluorochrome at the level of the individual phagosome. These frames are from a 45 minute movie. Flow cytometry (C) enables examination of the heterogeneity of the activity across the cell population at the level of each individual cell. Analysis by flow cytometry can be combined with immunofluorescence with antibodies against pathogens, or cell surface markers. Figure 2C Images courtesy of J. Enninga [Institut Pasteur, Paris].

FIG 3

FIG 3. The phenotypic differences between resting and activated macrophages are reflected in the physiology of their phagosomal compartments

The functions fulfilled by macrophages are modulated by their degree of stimulation, whether this is by exogenous mediators such as microbe-derived TLR agonists or endogenous activators such as cytokines and chemokines. Resting macrophages function in the absence of any inflammatory stimuli and their primary role is cleansing the body of cellular debris such as apoptotic cells. The phagosomal compartment of the resting macrophage is highly degradative for efficient processing of this material. In contrast, macrophages activated by exposure to TLR agonists or cytokines such as IFNγ, demonstrate alterations in several aspects of phagosome physiology. The superoxide burst is enhanced in its intensity (see also the Box 1 figure). Microbial killing in activated macrophages relies more heavily on the production of reactive oxygen and nitrogen intermediates. In addition, the phagolysosomal milieu of activated macrophages is actually less proteolytically active than that of a resting macrophage. This level of proteolysis is similar to that observed in dendritic cells, therefore, the implication is that this switch may enhance the half-life of epitopes and thereby maximize the antigen sampling and presenting capacity of activated macrophages.

Box Figure

Box Figure

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