Flow analysis of MHC molecules and other membrane proteins in isolated phagosomes

Abstract

A method was developed to apply flow cytometry analysis to the characterization of individual phagosomes. Macrophages were incubated with latex beads and homogenized to release the phagosomes. Intact cells and nuclei were removed by low speed centrifugation, and a crude phagosome preparation was fixed with paraformaldehyde. Distinct optical properties of latex bead phagosomes allowed their analytic isolation from other organelles and cell fragments by flow analysis using a narrow gate based on scatter parameters. Furthermore, separate gates were established for phagosomes containing one, two and even three beads, which were sorted and examined by electron microscopy (EM). EM showed that the phagosomal membrane was closely apposed to the latex bead in most phagosomes, but some more spacious phagosomes were also observed. Phagosomes were immunolabeled and subjected to flow analysis for MHC-I and MHC-II molecules and lysosomal membrane markers (LAMPs). The proportion of LAMP-positive phagosomes increased with incubation time, reflecting maturation of phagolysosomes. Significant staining for MHC-I and MHC-II was demonstrated and remained relatively constant with time. Flow analysis of phagosomes allows the characterization and comparison of individual phagosomes, and the identification of subpopulations of phagosomes with differing membrane compositions. It also provides the advantage of analytically isolating phagosomes from other components of the cell without the need for extensive prior physical purification. Thus, it can be used to rapidly assess changes in phagosomal membrane composition as a function of phagosome maturation.

Introduction

Certain cells, such as macrophages and polymorphonuclear leukocytes, are highly efficient in the uptake of particulate matter and microbes via phagocytosis (Silverstein et al., 1977; Allen and Aderem, 1996). Phagocytosis results in the production of membrane bound organelles called phagosomes, which are of plasma membrane origin. Phagosomes fuse with endosomes (Mayorga et al., 1991; Desjardins et al., 1994), and they eventually fuse with lysosomes to form phagolysosomes (Silverstein et al., 1977). Budding of phagosome-derived vesicles also allows the removal of membrane and lumenal material from phagosomes for recycling to the plasma membrane (Muller et al., 1980b) or transport to endosomes (Pitt et al., 1992) and possibly other intracellular organelles. Thus, membrane proteins and lumenal contents are both delivered to and removed from phagosomes in a series of membrane fusion and fission events. This allows sorting of phagosome-associated molecules and extensive modification of phagosomal composition with time (Desjardins et al., 1994; Desjardins, 1995), a process referred to as phagosomal maturation (Berón et al., 1995).

One consequence of phagocytosis is the processing of phagocytosed antigens and their presentation to T cells. Phagocytosed antigens are primarily processed via the MHC class II (MHC-II) antigen processing pathway (Ziegler and Unanue, 1981; Harding and Geuze, 1992; Pfeifer et al., 1992; Harding, 1995), and they can also be processed via recently discovered alternate MHC class I (MHC-I) antigen processing mechanisms (Harding, 1995; Rock, 1996). Phagocytosed antigens are proteolytically degraded within phagosomes or phagolysosomes, and antigen-derived peptides subsequently bind to MHC molecules, but the precise roles of phagosomes in antigen processing have not been clearly defined. Even the levels of MHC molecules in phagosomes remain poorly defined. Several reports have identified MHC molecules in phagolysosomes, largely by immuno-electron microscopy (immuno-EM) (Antoine et al., 1991; Lang and Kaye, 1991; Harding and Geuze, 1992), but studies in other systems have suggested that MHC molecules may be largely excluded from phagosomes (Clemens and Horwitz, 1992, Clemens and Horwitz, 1993).

Several techniques have been used to analyze the composition of phagosomes. Seminal studies by Muller et al. used a technique of in situ labeling of phagosomal/phagolysosomal membrane proteins for subsequent biochemical analysis (Muller et al., 1980a, Muller et al., 1980b, Muller et al., 1983). Other studies have involved the purification of phagosomes by differential centrifugation, density gradient centrifugation or organelle electrophoresis (Hasan et al., 1997), and characterization of phagosomal membrane proteins by Western blotting (Desjardins et al., 1994). Phagosomal maturation has been studied by fluorescence microscopy of cells after phagocytic challenge (Allen and Aderem, 1995; Oh et al., 1996). Immuno-electron microscopy (immuno-EM) has also been used by several groups to characterize the level of specific markers in phagosomal membranes and the interactions of phagosomes with other intracellular organelles (Antoine et al., 1991; Lang and Kaye, 1991; Harding and Geuze, 1992; Rabinowitz et al., 1992; Desjardins et al., 1994). Despite the availability of these approaches, a new method that would allow rapid quantitative assessment of the composition of individual phagosomes would facilitate future studies of phagosome composition and maturation.

We have developed a technique to rapidly characterize individual latex bead phagosomes and analyze their composition and maturation. This technique utilizes the ability of the flow cytometer to analytically separate phagosomes from all other organelles and whole cells, thereby allowing analysis of each individual phagosome without extensive prior physical purification. Phagosomes derived from bone marrow and peritoneal macrophages were stained for MHC molecules and lysosome-associated membrane proteins (LAMPs), and subjected to flow analysis. This allowed the quantitative assessment of changes in membrane markers at different time points as a function of phagosomal maturation.