Endocytosis & phagocytosis

A scientist is someone who has made all the mistakes there are; but in a very narrow field Niels Bohr

Phagocytosis showing extreme macrophage cytoplasmic streaming round an opsonised droplet
Whilst working on secretion, I became interested in the recovery of plasma membrane and membrane receptors by endocytosis as well as the extreme instance of phagocytosis. I wanted to understand more about the receptors, endocytosis triggering and traffic of plasma membrane internalisation. Since my own research efforts, endocytosis and especially phagocytosis have become key areas of study in the area of host defence and repair. These range from the innate immune system to the killing of pathogenic micro-organisms and clearance of apoptotic cells.

My work focused on several related areas: 1. The uptake and killing of bacteria and yeasts by neutrophils and macrophages 2. pH changes in endosomes and phagosomes and 3. Attempts to investigate mechanisms by investigating the mechanisms of inhibitors of phagosome - lysosome fusion and in vitro experiments to study fusion of isolated lysosomes with micro-organism containing phagosomes. In addition I measured pH changes in endosomes and phagocytic vacuoles and in measured in vivo phagosome pH changes and during the uptake of small molecules by receptor-mediated endocytosis.

Dynamic measurement of endosome and phagosome pH

Flow cell to measure cell pH
Quartz fluorimeter cuvette and perfusable coverslip assembly for measuring intracellular pH

Left image shows the set-up for measuring the pH of endocytotic vesicles. Typically a pH indicator: fluorescein isothiocyanate (FITC) was conjugated to bacteria, yeasts or antibodies and incubated with target leucocytes attached to a glass coverslip. After washing, the coverslip was mounted in the holder shown then perfused by temperature - controlled PBS in a quartz cuvette within a sensitive fluorescence spectrometer. The fluorescence spectrum of the probe was continuously recorded during endocytosis. Numerous controls including other pH sensitive and insensitive fluorophores were included to validate the system. Right hand image shows the emission spectra of endocytosed fluorescein conjugate. The lower spectrum (a) represents intracellular pH 5 The top spectrum (b) was obtained after adding trace monensin to collapse transmembrane pH gradients and corresponds to the pH of the extracellular medium: pH 7.4

Emission spectra of FITC-dextran conjugate taken up by a monolayer of macrophages

Fluorescence micrograph of labelled yeast phagocytosed by mouse macrophages

Left picture shows a fluorescence microscope image of yeast opsonised by fluorescein-labelled S. cerevisiae within mouse macrophage phagosomes. The normal result was a rapid reduction of pH as the phagosome was acidified. In the electron micrograph (right) lysosomes can be seen fusing with a yeast containing phagosome membrane. Note the close apposition of the phagosome membrane to the yeast. Which macrophage plasma membranes are internalised / recycled during phagocytosis and what membrane proteins are inserted and which cytoplasmic proteins are associated has been extensively studied in many forms of endocytosis, but much remains to be discovered!

Electron micrograph showing phagosome - lysosome fusion in a mouse macrophage after phagocytosis of yeast

Respiratory burst of neutrophils is associated with a transient increase in phagosome pH

Using the above system I investigated the uptake of fluorescein-labelled Staphylococci by human neutrophils with Anthony Segal (University College, London). Phagocytosis and killing is accompanied by an increase in non-mitochondrial respiration (the respiratory burst). In neutrophils from patients with chronic granulomatous disease (CGD) this burst is absent and killing is impaired leading to chronic infections. CGD neutrophils lack a functional cytochrome b which is a component of the oxidase system responsible for the respiratory burst of normal cells

Using the pH sensing system described above we noted that in normal neutrophils, phagocytosed bacteria were exposed to a significant transient increase in vacuole pH, before the phagosome environment became acidic. By contrast this pH increase was absent in neutrophils from CGD patients. In this case, the phagosomes were acidified immediately after uptake. This pH increase appears to be a key stage in microbial killing. This transient pH increase in normal neutrophils was confirmed microscopically by staining yeasts prior to phagocytosis with the pH indicator dye bromothymol blue.

Although the difference in the pH dynamics of the phagosome in normal versus CGD neutrophils has been confirmed, the biochemistry of the phagosome remains a detailed area of research. The advances since our work in 1990 has been reviewed by Tony Segal (article in press) and make fascinating reading!

pH in the endosome

With W. Howard Evans at the MRC labs I studied pinocytosis and receptor-mediated endocytosis in mouse macrophages and BHK fibroblasts.using the conjugated fluorescein technique. Whether by pinocytosis (drinking) or receptor-mediated uptake, fluorescent label was transferred to acidic compartments (pH 5 - 5.5) within cells with a half-time of less than 4 minutes. In contrast, yeast, pre-opsonised by fluorescein-labelled antibody was exposed to a high pH environment (relative to the external medium) within 2 min; a finding similar to that seen in normal human neutrophils described above. In all these cases, proteolysis of endocytosed materials did not occur, indicating that fusion with lysosomes occurred at a later stage in both processes.

Since this work the pathways of endocytosis have been extensively studied and dissected into different stages requiring fusion between distinct organelles (Viz: early and late endosomes, recycling endosomes, lysosomes). pH measurements and their dynamics within these organelles have also been made more precise with special probes. Intracellular membrane traffic and the mechanisms of organelle formation,transport and targeting remains an area of very active research.

Clinical indications

  • Chronic Granulomatous disease

Current research

Since the coronavirus pandemic, this whole area of research has received intense activity which would be impossible to even summarise here as so much is ongoing. Sufficient to note that pathogen recognition & host response as well as intracellular trafficking have gain an unprecedented importance over the last few years. Further studies at all levels (Molecular to clinical) will certainly pay dividends for human healthcare.

References

  1. The respiratory burst of phagocytic cells is associated with a rise in vacuole pH. Segal A W, Geisow, M, Garcia, R, Harper, A &Miller, R. (1981) Nature 290 406-409
  2. Chemical inhibitors of phagosome-lysosome fusion in cultured macrophages also inhibit saltatory lysosomal movements. Hart P, Young M R Jordan M M Perkins, W J & Geisow, M J (1983) J. Exp. Med. 158 477-492
  3. pH in the endosome Michael J Geisow and W Howard Evans (1984) Experimental Cell Research 150 36-46
  4. Chemical inhibitors of phagosome-lysosome fusion in cultured macrophages also inhibit saltatory lysosomal movements. Hart P, Young M R Jordan M M Perkins, W J & Geisow, M J (1983) J. Exp. Med. 158 477-492
  5. Site of action of a polyanion inhibitor of phagosome-lysosome fusion in cultured macrophages. Geisow M J, Beaven, G H, Hart, P D'Arcy & Young, M R (1980) Experimental Cell Research 126 159-165
  6. Extracellular fusion of macrophage phagosomes with lysosomes. Michael Geisow, Philip D'Arcy Hart & M. Robert Young (1982) Cell Biology International Reports 6 361-367