Phagosomes are formed when phagocytic cells ingest particles such as bacteria, viruses or synthetic beads of different kinds. The environment within the phagosome gradually changes to generate degradative conditions. These changes require multiple interactions between the maturing phagosomes and the endocytic and the biosynthetic pathway. The phagosomes probably communicate with endocytic organelles by a transient fusion event, often referred to as the ‘kiss-and-run’ hypothesis. We have studied the role of endocytic organelles in the phagocytic pathway of J774 cells, a mouse macrophage cell line. We have used magnetic Dynabeads coated with 125ITC-IgG and 125ITC-OVA as phagocytic probes and were able to isolate the phagosomal fraction by means of a magnet. To separate lysosomes from other organelles in the endocytic pathway we allowed the cells to endocytose a pulse of colloidal gold particles complexed with ovalbumin. By combining this density shift technique with subcellular fractionation of a postnuclear supernatant in Percoll gradients we could isolate three endocytic fractions corresponding to early endosomes (the light Percoll fraction), late endosomes (the dense Percoll fraction) and lysosomes (the gold fraction). We observed that the proteins linked to the ingested beads are initially cleaved in the phagosomes. This cleavage is inhibited by leupeptin, a thiol-protease inhibitor, and requires an acidic environment. However, efficient communication between the phagosomes and the endocytic pathway leads to the transfer of dissociated phagocytosed peptides of different sizes to late endosomes and lysosomes for further processing. Consequently, the late endosomes and the lysosomes may be involved in the degradation of phagocytosed compounds.

REFERENCES

Aniento
F.
,
Gu
F.
,
Parton
R. G.
,
Gruenberg
J.
(
1996
).
An endosomal bCOP is involved in the pH-dependent formation of transport vesicles destined for late endosomes.
J. Cell Biol
133
,
29
41
Berg
T.
,
Kindberg
G. M.
,
Ford
T.
,
Blomhoff
R.
(
1985
).
Intracellular transport of asialoglycoproteins in rat hepatocytes.
Exp. Cell. Res
161
,
285
296
Berg
T.
,
Gjoen
T.
,
Bakke
O.
(
1995
).
Physiological functions of endosomal proteolysis.
Biochem. J
307
,
313
326
Desjardins
M.
,
Celis
J. E.
,
van Meer
G.
,
Dieplinger
H.
,
Jahraus
A.
,
Griffiths
G.
,
Huber
L. A.
(
1994
).
Molecular characterization of phagosomes.
J. Biol. Chem
269
,
32194
32200
Desjardins
M.
,
Huber
L. A.
,
Parton
R. G.
,
Griffiths
G.
(
1994
).
Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus.
J. Cell Biol
124
,
677
688
Harding
C. V.
,
Song
R.
(
1994
).
Phagocytic processing of exogenous particulate antigens by macrophages for presentation by class I MHC molecules.
J. Immunol
153
,
4925
4933
Horisberger
M.
,
Rosset
J.
(
1977
).
Colloidal gold, a useful marker for transmission and scanning electron microscopy.
J. Histochem. Cytochem
25
,
295
305
Kovacsovics-Bankowski
M.
,
Rock
K. L.
(
1995
).
A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules.
Science
267
,
243
246
Lang
T.
,
Hellio
R.
,
Kaye
P. M.
,
Antoine
J. C.
(
1994
).
Leishmania donovani-infected macrophages: characterization of the parasitophorous vacuole and potential role of this organelle in antigen presentation.
J. Cell Sci
107
,
2137
2150
Malaviya
R.
,
Twesten
N. J.
,
Ross
E. A.
,
Abraham
S. N.
,
Pfeifer
J. D.
(
1996
).
Mast cells process bacterial Ags through a phagocytic route for class I MHC presentation to T cells.
J. Immunol
156
,
1490
1496
Mayorga
L. S.
,
Bertini
F.
,
Stahl
P. D.
(
1991
).
Fusion of newly formed phagosomes with endosomes in intact cells and in a cell-free system.
J. Biol. Chem
266
,
6511
6517
Oh
Y. K.
,
Swanson
J. A.
(
1996
).
Different fates of phagocytosed particles after delivery into macrophage lysosomes.
J. Cell Biol
132
,
585
593
Pfeifer
J. D.
,
Wick
M. J.
,
Russell
D. G.
,
Normark
S. J.
,
Harding
C. V.
(
1992
).
Recombinant Escherichia coli express a defined, cytoplasmic epitope that is efficiently processed in macrophage phagolysosomes for class II MHC presentation to T lymphocytes.
J. Immunol
149
,
2576
2584
Pfeifer
J. D.
,
Wick
M. J.
,
Roberts
R. L.
,
Findlay
K.
,
Normark
S. J.
,
Harding
C. V.
(
1993
).
Phagocytic processing of bacterial antigens for class I MHC presentation to T cells.
Nature
361
,
359
362
Pitt
A.
,
Mayorga
L. S.
,
Schwartz
A. L.
,
Stahl
P. D.
(
1992
).
Transport of phagosomal components to an endosomal compartment.
J. Biol. Chem
267
,
126
132
Pitt
A.
,
Mayorga
L. S.
,
Stahl
P. D.
,
Schwartz
A. L.
(
1992
).
Alterations in the protein composition of maturing phagosomes.
J. Clin. Invest
90
,
1978
1983
Pittman
R. C.
,
Carew
T. E.
,
Glass
C. K.
,
Green
S. R.
,
Taylor
C. A.
Jr.
,
Attie
A. D.
(
1983
).
A radioiodinated, intracellularly trapped ligand for determining the sites of plasma protein degradation in vivo.
Biochem. J
212
,
791
800
Rabinowitz
S.
,
Horstmann
H.
,
Gordon
S.
,
Griffiths
G.
(
1992
).
Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages.
J. Cell Biol
116
,
95
112
Slot
J. W.
,
Geuze
H. J.
(
1985
).
A new method of preparing gold probes for multiple-labeling cytochemistry.
Eur. J. Cell Biol
38
,
87
93
Tardieux
I.
,
Webster
P.
,
Ravesloot
J.
,
Boron
W.
,
Lunn
J. A.
,
Heuser
J. E.
,
Andrews
N. W.
(
1992
).
Lysosome recruitment and fusion are early events required for Trypanosome invasion of mammalian cells.
Cell
71
,
1117
1130
Tjelle
T. E.
,
Brech
A.
,
Juvet
L. K.
,
Griffiths
G.
,
Berg
T.
(
1996
).
Isolation and characterization of early endosomes, late endosomes and terminal lysosomes: Their role in protein degradation.
J. Cell Sci
109
,
2905
2914
van Weert
A. W. M.
,
Dunn
K. W.
,
Geuze
H. J.
,
Maxfield
F. R.
,
Stoorvogel
W.
(
1995
).
Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump.
J. Cell Biol
130
,
821
834
Wang
Y. L.
,
Goren
M. B.
(
1987
).
Differential and sequential delivery of fluorescent lysosomal probes into phagosomes in mouse peritoneal macrophages.
J. Cell Biol
104
,
1749
1754
Wattiaux
R.
,
Jadot
M.
,
Dubois
F.
,
Wattiaux-de Coninck
S.
(
1996
).
Phagocytosis by rat liver: Relationships between phagosomes and lysosomes.
Biochem. Biophys. Res. Commun
220
,
569
574
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