Lysosomal Enzyme Trafficking between Phagosomes, Endosomes,
and Lysosomes in J774 Macrophages
ENRICHMENT OF CATHEPSIN H IN EARLY ENDOSOMES*
Volker
Claus
,
Andrea
Jahraus§,
Torunn
Tjelle¶,
Trond
Berg¶,
Heidrun
Kirschke
,
Heinz
Faulstich
, and
Gareth
Griffiths§
From the
Max Planck Institute for Medical Research,
Heidelberg, § EMBL, 69117 Heidelberg, Germany,
¶ Department of Biology, University of Oslo, Oslo, 0316 Norway,
and
Department of Biochemistry, University of Halle, D-06097
Halle, Germany
 |
ABSTRACT |
In this study we take advantage of recently
developed methods using J774 macrophages to prepare enriched fractions
of early endosomes, late endosomes, dense lysosomes, as well as
phagosomes of different ages enclosing 1-µm latex beads to
investigate the steady state distribution and trafficking of lysosomal
enzyme activity between these organelles. At steady state these cells appear to possess four different cellular structures, in addition to
phagolysosomes, where acid hydrolases were concentrated. The first site
of hydrolase concentration was the early endosomes, which contained the
bulk of the cellular cathepsin H. This enzyme was acquired by
phagosomes significantly faster than the other hydrolases tested. The
second distinct site of lysosomal enzyme concentration was the late
endosomes which contain the bulk of cathepsin S. The third and fourth
large pools of hydrolases were found in two functionally distinct types
of dense lysosomes, only one of which was found to be secreted in the
presence of chloroquine or bafilomycin. Among this secreted pool was
soluble furin, generally considered only as a membrane-bound
trans-Golgi network resident protein. Thus, the organelles usually
referred to as "lysosomes" in fact encompass a growing family of
highly dynamic but functionally distinct endocytic organelles.
 |
INTRODUCTION |
De Duve's (1) now classic studies of the "lysosome" led to
the concept that this membrane enclosed structure, visualized by the
electron microscopic studies of Novikoff and others, defined a single
type of acidic organelle that encloses all the mature acid hydrolases
in the cell. That this simple view has survived so long (see
e.g. Refs. 2 and 3) is surprising when one considers the
extensive data published since approximately 1980 showing that in many
cell types lysosomal enzymes at steady state are enriched in at least
two physically distinct organelles, the so-called late endosomes (low
density lysosomes or prelysosomes) and the structures now referred to
as lysosomes (4, 5). In human fibroblasts these two sets of organelles
were shown to divide the total cellular acid hydrolase pool equally
between them (6), although in most cell types investigated 80-90% is usually maintained in the dense lysosomes (4). The term lysosome is now
operationally defined as the kinetically most distal compartment of the
endocytic pathway that is relatively dense in fractionation studies and
which is devoid of recycling receptors such as the two mannose
6-phosphate receptors (see Refs. 4 and 7).
We have previously proposed that the late endosomes and the dense
lysosomes interact in some fashion such that the Lgp-Lamp family of
(lysosomal) membrane proteins are in similar concentrations in the
membranes of both organelles (8, 9). That the two sets of structures
equilibrate in some way is also argued by cell-cell fusion experiments
(11, 12) and by other in vivo data (13). Nevertheless, these
two organelles are distinct as seen by the presence of high
concentrations of the cation-independent mannose 6-phosphate receptor,
as well as Rab7 in the late endosomes, but not in lysosomes of cells
such as normal rat kidney or the presence of the regulatory subunit of
the cyclic AMP-dependent protein kinase in the late
endosomes (as well as other locations) but not in lysosomes of
Madin-Darby bovine kidney cells (14). In J774 macrophages, which we
used for this study, the two Rab proteins, Rab5 and Rab7, while mostly
depleted from lysosomes are enriched in both early and late endosomes
(15),1 a clear difference to
all the other cell types investigated (9). For these reasons we
emphasize that the organelles operationally referred to here as early
endosomes, late endosomes, and lysosomes from J774 cells may not all be
strictly equivalent to the organelles we and others have defined more
precisely in cells such as normal rat kidney, baby hamster kidney,
Madin-Darby canine kidney cells, or hippocampal neurons (9). However,
these fractions from J774 cells have been extensively characterized in
our previous studies (15).1
There is also considerable evidence that, in addition to late endosomes
and lysosomes, early endosomes can contain small but functionally
significant amounts of some lysosomal enzymes (see Ref. 5 for review).
The simplest way to rationalize this early endosome pool is that it
represents newly synthesized hydrolases that have been delivered in
clathrin-coated vesicles that originate from the trans-Golgi network
(16, 17). In the study by Ludwig et al. (16) significant
amounts of newly synthesized lysosomal enzymes were detected in the
early endosomes by both biochemical and electron microscopic
approaches. In the case of rabbit alveolar macrophages, Diment et
al. (18) could show that ~35% of the total cathepsin D activity
was associated with "light endosomal membranes"; significantly, a
fraction of this putative early endosome pool was at least transiently
membrane-bound, by a mannose 6-phosphate-independent mechanism,
suggesting the possibility of a specific and more residential association with the membranes of (early) endocytic organelles by an
unknown mechanism. Indeed, the idea of a mannose
6-phosphate-independent mechanism for keeping a number of newly
synthesized hydrolases membrane-bound at different stages in their
transport from the endoplasmic reticulum to the endocytic pathway is
also consistent with many other lines of evidence (e.g.
Refs. 18-21).
Biodegradable organisms such as bacteria, yeast, or protozoans as well
as non-degradable particles such as carbon, asbestos, or latex beads
can be taken up by phagocytosis, especially by macrophages and
neutrophils. In most cases, after their formation phagosomes can fuse
with elements of the endocytic pathway, a process enabling the transfer
of hydrolytic enzymes into the phagosome. The available evidence argues
that newly formed phagosomes can fuse with early endosomes
(22-25)1 as well as with later endocytic organelles
(26-30).1
In the present study we made use of a recently developed method using
J774 macrophages to prepare enriched fractions of operationally defined
early endosomes, late endosomes, as well as lysosomes that had
accumulated colloidal gold (15). In addition, we made use of our recent
approach to prepare highly purified fractions of phagosomes
(phagolysosomes) enclosing 1-µm latex beads (29). Most of the data in
this study deal with careful kinetic measurements of the parallel
activities of 10 mostly well known acid hydrolases in these different
organelles. The results argue that in J774 cells different acid
hydrolases are selectively enriched in at least four different types of
endocytic organelles at steady state.
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MATERIALS AND METHODS |
Cells--
J774 cells were cultured as described previously
(29). For experiments using horseradish peroxidase
(HRP)2 over 70% confluent
cells were incubated 30 min at 37 °C with 2 mg/ml HRP (type II,
Sigma) in Dulbecco's modified Eagle's medium supplemented with 1 mg/ml mannan (Sigma, Munich), followed by a chase incubation of 30 or
90 min in marker-free Dulbecco's modified Eagle's medium. For
measurement of the secretion (HRP and cellular enzymes), the cell
culture media were replaced, after a brief rinse with PBS, by 10 mM Hepes in PBS, with or without 180 µM chloroquine, and the cells were further incubated at 37 °C for the
indicated times. Extracellular enzyme activities were determined in the
cell culture supernatant after 5 min centrifugation at 1200 rpm. The
intracellular activity of HRP was measured after extensive working of
the cells with PBS at 4 °C in the lysate of harvested cells. In
these experiments no significant amounts of cell-associated HRP could
be detected when the cells were incubated under the same conditions at
4 °C (results not shown). For treatment with inhibitors, cells were
incubated in the different preincubation, pulse, and chase media
supplemented with 10 mM Hepes and either 100 nM
bafilomycin A1, 180 µM chloroquine, or 20 µg/ml
cycloheximide (all from Sigma, Munich).
Preparation of Endocytic Fraction and Phagosomes--
Late
endosomes and lysosomes were isolated from one set of macrophages. The
procedure was a modification of the method described by Tjelle et
al. (15). Briefly, for the preparation of late endosomes and
lysosomes, J774 cells were allowed to internalize ovalbumin-conjugated
gold particles (10 nm) for 3 h and chased overnight to trace the
bulk of gold into the lysosomal compartment. Cells with or without gold
were homogenized and centrifuged at 750 × g for 7 min.
For early endosomes the PNS were mixed 1:1 with 80% metrizamide in
PBS, and this mixture was loaded at the bottom of a SW40 tube and then
overlaid with 17% Percoll in homogenization buffer (HB-250
mM sucrose, 3 mM imidazote buffer, pH 7.4, 1 mM dithiothreitol) followed by a layer of 500 µl of HB.
After centrifugation at 56,000 × g for 30 min, the
early endosomes were collected from the top of the self-formed Percoll
gradient. Late endosomes and lysosomes were isolated from a different
set of macrophages using ovalbumin-conjugated gold particles (10 nm) to
achieve a density shift between late endosomes and lysosomes. The gold
was internalized into J774 for 3 h and chased overnight to trace
the bulk of gold into the lysosomal compartment. The cells were
homogenized and centrifuged at 750 × g for 7 min. The
gold-containing fraction was found in the nuclear pellet, whereas the
late endosome fraction stayed in the PNS. This PNS was layered onto
17% Percoll/HB with a 50% sucrose cushion underneath to eliminate
gold particles left over in the PNS. After centrifugation at
56,000 × g for 1 h, the late endosomes
accumulated as a band in the Percoll gradient close to the sucrose
cushion. The nuclear pellet with the gold-containing lysosomes was
resuspended in 17% Percoll/HB and layered onto a 64% sucrose cushion.
A centrifugation at 40,000 × g for 30 min separated
the gold-filled lysosomes, which pelleted through the sucrose cushion,
from the nuclei remaining on top of the gradient. The characterization
of these fractions is described by Tjelle et al. (15) and
Jahraus et al.1
It was shown by Tjelle et al. (15) and Jahraus et
al.1 that the transferrin receptor is enriched in the
early endosomal fraction but is essentially below detection in both the
late endosome and lysosome fractions. Moreover, the early endosome
specific marker EEAI (31) is detected only in the early endosome
fraction by Western blotting.1 In contrast cathepsin S is
highly enriched in the late endosome fraction.1. In
addition early and late endosome fractions could be distinguished by
kinetic studies with internalized horseradish peroxidase which forms a
plateau in the early and late endosomes after a 5-min pulse and a 5-min
pulse plus a 25-min chase, respectively. As for the late
endosomes/lysosomes the kinetics of HRP entry into the (isolated)
endocytic compartments varied slightly from experiment to experiment
(15).1 Although in some sets of experiments HRP reached the
late endosome 5-10 min before it reached lysosomes (15), the
difference was insignificant in others.1 Nevertheless these
two sets of organelles can be differentiated both by their structure
(15) and by their in vitro fusogenic properties.1
The preparation of phagosomes enclosing 1-µm latex beads was done
exactly as described by Desjardins et al. (29). The
quantification of phagosome number was done using a 10-100-fold
dilution by measurement of A at 600 nm. For the standard
curve, latex bead solution with different number of beads was
determined by counting them in a hemocytometer. The determination of
protein was done by the BCA-Microassay (Pierce).
Preparation of Membranes--
Early endosomal vesicles or PNS
were lysed by 10-fold dilution of the fraction in 5 mM
carbonate plus 150 mM NaCl at pH 11 or in 5 mM
phosphate plus 150 mM NaCl at pH 7, supplemented with either 0.5% Triton X-100 or 0.5% saponin, or, according to the method
of Ref. 32, by dilution in hypotonic 5 mM phosphate at pH 7 followed by 8 strokes through a 0.4 × 30 needle, followed by
incubation in 0.3 or 1 M NaCl or in 5 mM
mannose 6-phosphate (Sigma, Munich). The samples were incubated under
the different conditions for 30 min at 4 °C before the membranes
were pelleted by centrifugation for 40 min at 100,000 × g and 4 °C in a Ti 70 rotor (Beckman). The enzyme
activities were measured in the supernatant before and after the
centrifugation as described below.
Enzyme Assays--
HRP activities were determined at 37 °C,
using as substrate 1,2-phenylenediamine (Sigma, Munich) in a
concentration of 1 mg/ml in citric acid/phosphate buffer plus 0.13%
hydrogen peroxide, and 1 N sulfuric acid was used as a stop
reagent. Quantification was done spectrometrically via measurement at
490 nm. All cellular enzyme activities were measured fluorometrically
after pretreatment of the cellular fractions with 0.2% Triton X-100
for 20 min at 4 °C. The assays were performed at 37 °C with a
substrate concentration of 0.1-1 mM using specific
substrates, buffers, and pH for the different enzymes as listed in
Table I with the exception of cathepsin S
which was determined as described by Kirschke and Wiederanders (33). In
the case of cathepsin H some of the assays were also done in the
presence of 1 µM puromycin to exclude the presence of
aminopeptidase activity which also cleaves Arg-AMC (34). It should be
noted that since the substrate used to detect cathepsin L
(Z-Phe-Arg-AMC) may also react with cathepsin B (34), we refer to this
activity as cathepsin B + L. In contrast the substrate used to detect
cathepsin B (Z-Arg-Arg-AMC) is specific for this enzyme (35).
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Table I
List of substrates, buffers, and pH values used for the determination
of enzyme activities
The abbreviations used are: AMC, 7-amino-4-methylcoumarin; DPP II,
dipeptidyl peptidase II; MU, 4-methylumbelliferyl; Pyr,
L-pyroglutamic acid; Z, benzoyloxycarbonyl.
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In the case of the 4-methylumbelliferyl-derived substrates (Sigma,
Munich, or Calbiochem, Bad Soden) the quantification of fluorochromes
was done using an excitation (Ex) wavelength of 364 nm and emission
(Em) at 448 nm in a 10-fold dilution of 0.1 M acetate at pH
10.6 in the case of the 7-amino-4-methylcoumarin (AMC)-derived
substrates (all from Bachem, Heidelberg) at
Ex370 nm/Em460 nm in a 10-fold dilution of
0.1 M monochloroacetic acid, 0.1 M acetate at
pH 4.3. The amounts of enzyme activities were expressed as units, where
1 unit is the amount of enzyme that cleaves 1 nmol of substrate per
min.
Immunoelectron Microscopy--
J774 macrophages were allowed to
internalize 16 nm gold/BSA conjugates for 2 h at 37 °C followed
by an overnight chase in medium free of gold. They were subsequently
fed 5 nm gold/BSA for 5 min to fill early endosomes. The cells were
removed with proteinase K fixed with 4% formaldehyde and cryosections
prepared which were labeled with rabbit anti-rat cathepsin H (36)
followed by 10 nm gold-protein A. Profiles of early endosomes
(containing at least one 5-nm gold particle) and late endocytic
structures (containing at least one 16-nm gold particle) were
systematically sampled and photographed. The density of labeling for
cathepsin H over early endosomes, late endocytic structure, and the
nucleus control was carried out as described by Griffiths (10) using a
systematic sampling procedure.
Enzyme-linked Immunosorbent Assay--
The vesicular fractions
were lysed by 10 cycles of thawing and freezing, followed by 10 strokes
through a 0.4 × 30 needle. After centrifugation for 5 min at
13,000 rpm in an Eppendorf centrifuge to remove the gold,
i.e. in the lysosomal fraction, different amounts of the
supernatants, containing 1-3 µg of protein, were coated on
microtiter plates (Greiner, Nürtinpen) by incubation in 0.44%
carbonate buffer overnight at 4 °C. As a standard, different amounts
(2-120 ng) of human liver cathepsin H (Sigma, Munich) were coated in
the same way. Blocking was done by incubation with 1% BSA in PBS,
0.1% Tween 20 for 1.5 h at 37 °C. For detection we used as
primary antibody the rabbit anti-rat cathepsin H antibody, described
above, in a concentration of 9 µg/ml in PBS, 0.1% BSA. As a second
antibody we used a goat anti-rabbit IgG antibody conjugated with HRP
(Bio-Rad, Munich) in a working dilution of 1:500 in PBS. The incubation
time for each antibody was 1 h at 37 °C. The quantification of
bound HRP activity was carried out as described above.
 |
RESULTS |
Distribution of Acid Hydrolases in Endocytic Organelles--
By
using the method developed by Tjelle et al. (15) we prepared
enriched fractions of the three different endocytic compartments in
J774 cells, namely early endosomes, late endosomes, and lysosomes. The
rationale for this approach is to allow cells to internalize ovalbumin-coated gold particles (10 nm) which were then chased overnight into late endocytic structures. As seen by electron microscopy the bulk of this gold is found in large aggregates in mostly
spherical vesicles (lysosomes), whereas low levels of gold are present
also in other more heterogeneous structures which we define as late
endosomes (see Fig. 3 and Ref. 9). Following homogenization the bulk of
the gold-containing organelles pelleted at low speed with the nuclear
fraction; from this the lysosomal fraction could be purified using a
second centrifugation on a Percoll gradient. The PNS from this
preparation was used to enrich the late endosomes, and the PNS from a
separate set of cells was used to prepare the early endosome fraction.
For details of the characterization of these three different fractions,
see "Materials and Methods."
When the three endocytic fractions were assayed for the activities of
11 different acid hydrolases, 9 behaved as expected with the bulk of
activities in the lysosomal fraction. The rest of the activities of
these nine enzymes was distributed as expected from earlier studies (4)
with more in the late endosome fraction than in the early endosomes
(Fig. 1). There were three exceptions to
this general pattern. The first was
-hexosaminidase whose activity
in the early endosome fraction was significantly higher than that found
in the late endosomes. The second and most striking exception to the
general pattern was cathepsin H in that the bulk of the activity
(~70%) of this cysteine protease was restricted to the early
endosome fraction (Fig. 1). Since cathepsin H is known to have both
endopeptidase and aminopeptidase activity (35), we wanted to rule out
the possibility that we were assaying an aminopeptidase activity. The
activities of cathepsin H in all subcellular fractions were found to be
insensitive to 1 µM puromycin (data not shown), so that a
contribution of cellular aminopeptidases (34, 37) could be excluded.
Since the activity of this enzyme in the early endosome fraction was
not significantly affected by treatment of the cells for up to 3 h
with 20 µg/ml cycloheximide before isolation of the fractions
(results not shown) it appears that, for the most part, cathepsin H in
the early endosome pool is probably resident rather than a transient
passenger.

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Fig. 1.
Distribution of specific enzyme activities in
early endosomal (EE), late endosomal (LE), and
lysosomal (LYS) fraction of J774 cells. The endocytic
vesicles were isolated, and the enzyme activities were measured as
described under "Materials and Methods." For each activity the
total of the specific activities found in all vesicle fractions was set
at 100%. It should be noted that since the substrate used to detect
cathepsin L (Z-Phe-Arg-AMC) may also react with cathepsin B (35), we
refer to this activity as cathepsin B + L. In contrast the substrate
used to detect cathepsin B (Z-Arg-Arg-AMC) is specific for this enzyme
(35).
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After the experimental part of this project was completed, we found in
a parallel study1 that cathepsin S was highly enriched in
the late endosome fraction by immunoblot analysis. We therefore
analyzed the activity of this protease in the three fractions. As seen
in Fig. 1 also the activity of this enzyme is significantly higher in
the late endosome fraction, although the magnitude of the increase in
the late endosomes over the lysosomes was significantly less than that
seen by immunoblot analysis.
Cathepsin H in Early Endosomes--
Since the activity for
cathepsin H was found to be enriched in early endosomes, we decided to
investigate the location of this protein using a rabbit antibody
against rat cathepsin H that has previously been extensively
characterized (36). This antibody is specific for cathepsin H since it
neither recognizes the closely related cathepsins B, L, or D nor the
closely related plant protease papain (34). Since attempts to identify
cathepsin H by immunoblotting were not successful, we set up a
quantitative enzyme-linked immunosorbent assay to determine the amount
of cathepsin H from each endocytic fraction adsorbed to microtiter
wells. Purified cathepsin H was used to quantify the reaction, using a
HRP-based detection system. As shown in Fig.
2 the relative concentrations of
cathepsin H in the early endosome and lysosome fractions by this
approach was similar to that seen by enzymatic activity (Fig. 2).

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Fig. 2.
Quantitation of cathepsin H by enzyme-linked
immunosorbent assay in the different endocytic fractions of J774.
The estimation of the amount of cathepsin H was done using as a
standard human liver cathepsin H. The values shown represent the
mean ± S.E. from four experiments.
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We next looked at the distribution of cathepsin H using immunogold EM.
For this, we used thawed cryosections of J774 cells that had
internalized 16 nm gold to label late endocytic structures and 5 nm
gold (5 min) to label early endosomes. These sections were labeled with
the rabbit anti-cathepsin H antibody followed by protein A-gold (Fig.
3). The bulk of the labeling was detected in early endosomes, and only low levels were detected in 16 nm gold-filled organelles (mostly lysosomes). Although the overall extent
of the labeling on these thin cryosections was low, a quantitative analysis confirmed that the early endosome labeling was significantly higher than that on late endosomes/lysosomes as well as that over the
nuclear matrix (background) (Fig. 4).

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Fig. 3.
Cryosection of J774 macrophages that had
internalized 16 nm gold-BSA for 2 h followed by an overnight chase
and 5 nm gold-BSA for 5 min. The sections were labeled with
anti-cathepsin H and protein A-gold (10 nm). The arrowheads
in A and B indicate cathepsin H labeling over the
early endosome, whereas the arrows in B point to
two 10-nm gold particles over organelles which cannot be identified. In
this example the 16-nm gold-labeled structure (large
arrowheads indicate the 16 nm gold) is devoid of cathepsin H
labeling. P, plasma membrane. Bars, 100 nm.
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Fig. 4.
Quantification of cathepsin H labeling from
the experiment shown in Fig. 3. Labeling is expressed as
gold/µm2 over the nucleus (N), late endocytic
structures (LE), and early endosomes (EE).
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Since the above data showed that in J774 macrophages the bulk of
cathepsin H protein and activity was localized to early endosomes, we
performed some preliminary experiments to gain insights into the
mechanisms by which this "soluble" enzyme can be retained in a
dynamic organelle with a high flux rate of both fluid and membrane
components. For this, we took advantage of the procedure of Authier
et al. (32) in which the membrane fraction of interest is
hypotonically disrupted, followed by the determination of enzyme activities in the supernatant before and after high speed
centrifugation. Following preparation of the early endosomal vesicles
under isotonic conditions without homogenization, only
25% of the
cathepsin H activity was recovered in the supernatant (Fig.
5A). This level of "free"
cathepsin H was not significantly affected by homogenization under
hypotonic conditions followed by incubation in 0.3-1 M
NaCl, whereas a small but insignificant increase in the extent of
solubilization was seen following incubation with 5 mM
mannose 6-phosphate. The supernatant pool approached 50% with 0.5%
saponin. The only treatments we found to solubilize the bulk of
cathepsin H activity were 0.5% Triton X-100 or a pH 11 treatment (Fig.
5A) (which was also found to inhibit activity by
70%;
results not shown). Collectively, these data suggest that the bulk of
cathepsin H is maintained in the early endosome by a mannose
6-phosphate-independent mechanism, perhaps as a peripheral (lumenal)
membrane protein.

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Fig. 5.
Percentage of soluble activity of cathepsin H
(A) and furin (B) in subcellular fractions of
J774 cells. Cathepsin H activity was determined in early endosomal
vesicles and furin activity in the postnuclear supernatant, both before
and after high speed centrifugation of the total membranes. Before
pelleting the membranes the subcellular fractions were diluted either
in isotonic buffer or in a hypotonic buffer followed by homogenization
and incubation at the indicated conditions for 30 min at 4 °C. The
values shown represent the mean ± S.E. of three experiments. *,
the total of cathepsin H activity at pH 11 was only 30% that found at
pH 7. **, under these conditions no furin activity could be
detected.
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The Bulk of Furin Activity Is Soluble--
We were surprised to
see furin activity behaving in these experiments as a typical lysosomal
enzyme since furin is normally considered a trans-Golgi
network-spanning membrane protein (38). We therefore investigated the
fraction of this activity which was soluble and that which was
membrane-bound. For this we carried out the same procedure as for
cathepsin H. As shown in Fig. 5B under isotonic conditions
25% of furin activity was soluble. Following hypotonic conditions,
however,
80% of the activity of this enzyme became soluble.
Transfer of Acid Hydrolases to Phagosomes--
Phagosomes are
newly formed particles enclosing vesicles that are initially devoid of
acid hydrolases but, following a delay, acquire a full complement of
these enzymes following fusion with the organelles traditionally
referred to as lysosomes. Since our latex bead phagosomes can be highly
purified from cells at any stage after their internalization, we
decided to investigate the kinetics of acquisition of acid hydrolases
by phagosomes and compare these data with our previous experiments that
investigated transfer of internalized HRP and the Lamp membrane
proteins into phagosomes (29). For this, the enzyme activities were
estimated in purified phagosomes prepared at different times after
latex internalization. As shown in Fig. 6
it took between 4 and 8 h after the addition of the beads to cells
in order for the isolated phagosomes to acquire their full complement
of most of the hydrolases tested. The most striking exception was again
cathepsin H which was most rapidly acquired by latex phagosomes; the
activity of this enzyme measured at the earliest time point (20 min)
was the same as that seen at the latest time point (1-h pulse followed
by a 24-h chase). We saw a consistent drop in the activity of cathepsin
H at the 2-4-h time point (Fig. 6) whose significance is unclear. A
quantitation of the percentage of total cell activities of the various
hydrolases in phagosomes is shown in
Table II. The relative amounts of these enzymes analyzed in 4-h phagosomes range from 1.4% (cathepsin H) to
30% (cathepsin B + L) of the total cellular pool of these enzyme
activities (Table II).

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Fig. 6.
Enzyme activities of phagosomes of J774 cells
at different times after formation of the phagosomes. In case of
the 20-min time point cells were incubated for 20 min with latex beads,
while for the later time points cells were incubated with a pulse of
beads for 1 h followed by further incubation in medium free of
beads until the indicated time points. Phagosomes were isolated, and
the enzyme assays were performed. Values shown represent the mean ± S.E. of five experiments and are expressed relative to a fixed
number (1010) of latex beads.
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Table II
Details of enzyme activities in phagosomes of J774 cells
The isolation of phagosomes were done after 1-h pulse plus 3-h chase
incubation of cells with latex beads as described under "Material and
Methods." The enzyme activities were determined in the PNS and in the
phagosomal fraction, and the phagosomal associated activity was
correlated to the total activity in the PNS, which was set as 100%.
The values shown are mean ± S.D. of three different experiments.
DPP, dipeptidyl peptidase.
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A closer investigation of Fig. 6 showed other interesting patterns.
Although it took only 2 h for phagosomes to obtain their full
complement of
-mannosidase, it took 24 h for them to reach their peak concentration of
-glucosidase. Noteworthy, however, the
remaining seven hydrolases reached the phagosomes with essentially identical kinetics, reaching a plateau at 4-8 h. These results can be
contrasted with the
1 h for equilibration of the fluid marker HRP
between phagosomes and late endocytic structures and compared with the
6-8 h needed for late endosomes and lysosome-derived Lamp proteins
to reach their plateau level in phagosomes
(29).3
Effect of Chloroquine and Bafilomycin A1--
Many earlier studies
in the literature have shown that acidotropic amine reagents such as
chloroquine or ammonium chloride can have significant and often complex
effects on phagosome and endosome functions. Among these effects are a
neutralization of the low pH of the lumen of endocytic organelles (39)
as well as various effects on phagosome-endosome or phagosome-lysosome fusion (22, 27). We therefore investigated the effects of chloroquine,
as well as the more specific vacuolar proton ATPase inhibitor
bafilomycin A1 (40), on the process of lysosomal enzyme acquisition by
phagosomes.
When latex bead uptake was allowed to occur in the continued presence
of either 180 µM chloroquine or 100 nM
bafilomycin A1, there was a complete block of acquisition by phagosomes
of the activities of six out of the eight hydrolases tested. In the
case of
-hexosaminidase the block in delivery was of the order of 80%. In contrast, the delivery of cathepsin H was enhanced by both
drugs (Fig. 7). The simplest
interpretation of these unexpected results was that raising the lumenal
pH of cell vacuolar organelles leads to a block in fusion between
phagosomes and late endosomes but an enhanced rate of fusion of
phagosomes with the early endosomes, where the bulk of cathepsin H
resides.

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Fig. 7.
Enzyme activities of phagosomes isolated from
control J774 cells ( ) compared with phagosomes isolated from cells
treated with 180 µM chloroquine ( ) or 100 nM bafilomycin A1 ( ). The drugs were added to the
medium of the cells 1 h prior to the initiation of phagosome
formation and maintained up to the isolation of the phagosomes. Values
shown represent the mean ± S.E. of three experiments.
|
|
We next investigated whether chloroquine or bafilomycin A1 led to any
depletion of the hydrolases from endocytic compartments that would
normally deliver them to the phagosomes. Strikingly, under these
conditions, we saw a complete depletion from the cells of three of the
eight hydrolases tested (cathepsin B, B + L, and furin) from the cells
(Fig. 8A). In addition, the
activity of
-glucuronidase was diminished by about 50% (Fig.
8A). At the same time the activities of four other enzymes
(cathepsin H,
-galactosidase, DPPII, and
-hexosaminidase)
remained essentially unchanged (Fig. 8A). That the
activities of cathepsin B, B + L, and furin were indeed depleted from
the cells was shown directly by experiments showing that these
activities were released into the medium of living cells by a process
that was enhanced by chloroquine (Fig. 8B). Besides this
first group of hydrolases which were quantitatively secreted in the
presence of acidotropic drugs (and therefore could not arrive in
phagosomes), a second group of enzymes existed that showed no
significant secretion (Fig. 8B) but, likewise, did not reach
the phagosomes (Fig. 7). The most prominent examples of the latter were
DPPII,
-glucuronidase, and
-hexosaminidase. This experiment
provides evidence for different pools of lysosomal enzymes that are
differentially affected by bafilomycin and chloroquine. Collectively,
these results argue that in the presence of chloroquine or bafilomycin
the bulk of cathepsins B, B + L, and furin that would normally be
targeted to newly synthesized phagosomes became secreted, whereas the
other hydrolases remained in the cell in a pool that was now
inaccessible to phagosomes.

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Fig. 8.
Effect of acidotropic drugs on the secretion
of hydrolases. A, the enzyme activities were measured in the
postnuclear supernatant of cells, incubated for the indicated times
with either 180 µM chloroquine (Chl) or 100 nM bafilomycin A1 (Baf) prior to the isolation
of the PNS. The enzyme activities were calculated relative to the total
protein in the PNS and are shown as percentages of the values found in
the PNS of untreated cells (set at 100%). B, enzyme
activities in the supernatant of untreated J774 cell cultures ( ) or
cells treated with chloroquine ( ). At the 0 time point the cell
culture medium was replaced by PBS, 10 mM Hepes with or
without 180 µM chloroquine, and the cells were further
incubated at 37 °C (---) or 4 °C (- - -). At the indicated
times aliquots of the cell culture supernatant were removed, and after
low speed centrifugation the enzyme activities were determined in the
supernatant. Values represents the means ± S.E. of three
experiments.
|
|
We next asked whether chloroquine would have any effect on the
retention of hydrolases already acquired by phagosomes. For this, J774
cells were allowed to internalize beads for 1 h at 37 °C
followed by 1 h of chase. They were then treated with chloroquine and incubated for an additional 1 or 2 h at 37 or 4 °C before isolating the phagosomes and analyzing six hydrolases. As shown in Fig.
9, cathepsins B, B + L, and furin behaved
identically in that their enzyme activities were selectively
transferred out of phagosomes within 1 h after adding the
chloroquine. When the temperature was lowered from 37 to 4 °C, this
effect disappeared. In contrast, at 37 °C DPPII,
-hexosaminidase
and
-glucuronidase were depleted from phagosomes at a significantly
lower rate, being equivalent only to the levels seen for the three
other more mobile enzymes at the low temperature. This argues that
during phagosome fusions different classes of lysosomal enzymes can be
sorted, not only into but also out of phagosomes at different
rates.

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Fig. 9.
Effect of chloroquine on the retention of
hydrolases by phagosomes. J774 cells were allowed to internalize
latex beads at 37 °C for a 1-h pulse plus a 1-h chase, to accumulate
hydrolases in phagosomes, and then 180 µM chloroquine was
added at the 2-h time point (arrows). After addition,
phagosomes were isolated immediately or cells were incubated for a
further 1 or 2 h at 37 °C ( ) or at 4 °C ( ), before
isolation of the phagosomes. The enzyme activities were then
determined. Values shown represent the means ± S.E. of three
experiments.
|
|
Secretion of a Fluid Phase Marker--
Evidently, under different
conditions significant amounts of some acid hydrolases can be secreted
by J774 macrophages. The fact that this secretion was unaffected by a
4-h treatment of cycloheximide (data not shown) as well as the fact
that in the presence of chloroquine essentially all the cell-associated
activities of some enzymes could be secreted (see Fig. 8) suggested
that the secreted pool must originate from both late endosomes and lysosomes. In that case, it was relevant to ask whether a bulk endocytic marker internalized by J774 cells could also be secreted and
whether that secretion could be modulated by chloroquine.
For this, J774 cells were pulsed with HRP (in the presence of yeast
mannan to prevent binding to the macrophage mannose receptor) for 30 min at 37 °C and then chased for either 30 or 90 min at the same
temperature. Subsequently, the cells were either treated with
chloroquine for a further 60 min or left untreated. After this time the
amount of HRP found in the medium or remaining cell-associated was
estimated. As seen in Fig.
10A, after a 30-min pulse
and an additional chase period of 90 min there was a secretion of
25% of the total cell-associated HRP, a value that was not
significantly increased in the presence of chloroquine for the last
hour chase. An increase in the chase time to 150 min did not affect the
amount of HRP secreted in the absence of chloroquine. However, when
chloroquine was added for the last 60 min of this latter condition the
bulk (58%) of the cellular HRP was now secreted into the medium (Fig. 10B). This shows that, in addition to their unusually high
level of secretion of a fluid phase internalized marker from late
endocytic compartments, J774 cells appear to possess a late endocytic
organelle, presumably a lysosome, that is significantly accessible to
HRP only after a 120-180-min internalization period and which appears to have all the hallmarks of a regulated secretory vesicle.

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Fig. 10.
Effect of chloroquine on the secretion of
HRP in J774 cells. HRP activity was quantified in the
extracellular supernatant (filled columns, secreted pools)
or the cell pellet (open columns, cell-associated). The
values shown represent the means ± S.E. of three experiments in
which cells were allowed to internalize the fluid phase marker HRP for
a 30-min pulse followed by a chase of 90 min (A) or 150 min
(B) in marker-free medium. At the end of these chase
periods, the cell culture media were replaced by 10 mM
Hepes/PBS, with or without 180 µM chloroquine, and the
cells were incubated for a further 60 min.
|
|
 |
DISCUSSION |
Cathepsin H in Early Endosomes--
A novel observation from our
study was the finding that cathepsin H is enriched in early endosomes
of J774 macrophages, as determined by its enzymatic activity, by
protein determination, and by immuno-EM. This cysteine protease thus
appears to be the first example of an acid hydrolase that is more
concentrated in early endosomes than in all other endocytic
compartments. The information that is available about this enzyme (it
is predominantly an aminopeptidase as well as an endopeptidase with a
pH optimum of
6.5 (33, 41, 42)) fits well with estimations of the pH
in the early endosomes (43, 44). This enzyme is found in a relatively
high concentration in a number of tissues such as kidney and spleen
and, significantly with respect to the present study, also in
macrophages (45). Although J774 macrophages possess detectable levels
of almost all the hydrolases analyzed in their early endosomes, a
finding consistent with many earlier studies (5), cathepsin H is the
first example of a vacuolar hydrolase whose activity is the highest in
the most proximal endocytic compartment. The precise mechanism by which
the cathepsin is retained in the early endosomes must await a more
detailed study.
Fusion of Phagosomes with Endocytic Compartments in Vivo--
It
has long been known that phagosomes acquire a full complement of
lysosomal acid hydrolases during their interactions with endocytic
organelles. It has also been well established that this mixing of
contents is a consequence of fusion events (26, 29, 46-48). We have
recently shown that latex bead phagosomes fuse in vitro in
an age-dependent fashion with enriched fraction of early
endosomes, late endosomes, and lysosomes, prepared as in the present
study.1 In that study phagosomes until
5-7 h of age
were fusogenic with early and late endosomes, whereas their fusogenic
life with lysosomes was extended to
13 h. After this time phagosomes
lost their capability to fuse in vitro.1.
In vivo EM studies confirmed that fusion of 24-h phagosomes with gold-filled endocytic organelles is significantly reduced, although a low level of content mixing could be detected using an
HRP-based assay (49).
It is striking that the length of the high fusion-competent state of
phagosomes in the in vitro study1 coincides very
well with the times needed here for 7 of the 10 acid hydrolases we
tested to reach their steady states in phagosomes. In other words
phagosomes appear to be switched to the "low fusion" state (4-13
h; depending on the target) only after they have received a full
complement of these enzymes (4-8 h). By this reasoning it is
interesting to note that most of the phagosomal hydrolases probably
come from lysosomes since, at least in vitro, the fusion of
phagosomes with both early and late endosomes is turned off before the
phagosomes have acquired their full capacity of acid hydrolases.
Moreover, the lysosome fraction is the richest source of most of the
hydrolases of interest. Interestingly, the in vitro active
fusogenic life of the latex phagosomes with lysosomes (13 h) exceeds
the
8 h needed to transfer essentially the full complement of acid
hydrolases, as well as the Lamp proteins (29), to the newly formed
phagosomes in vivo. That the fusion condition after 13 h is "low" rather than "off" (49) is also supported by our finding here that the phagosomes acquire their full complement of
-glucosidase only after 24 h; a low level of fusion must be continuing for this time. Thus, this enzyme is somehow selectively retained in the lumen of late endocytic organelles.
The acquisition of a full complement of cathepsin H activity within 20 min of phagosome internalization supports earlier data showing that
phagosomes can fuse avidly with early endosomes in vivo (30,
25) as they do in vitro (23).1 An effective
block in the acquisition by phagosomes of the bulk of the other nine
hydrolases tested, mostly acquired from late endosomes and lysosomes,
was seen in the presence of chloroquine and bafilomycin A1. In
contrast, the activity of cathepsin H in phagosomes was not decreased
but rather seemed to be increased by these drugs. We interpret these
results to indicate that a pH below neutrality needs to be maintained
in at least one of the partners in the proposed fusion between
phagosomes and late endosomes or lysosomes, but it is clearly not
essential for the early presumed fusion of phagosomes with early
endosomes. Although the molecular details are unclear the idea emerges
that by tampering with pH (as well as perhaps other factors), these
acidotropic drugs can inhibit some membrane transport processes (late
endosomes/lysosomes to phagosomes) while stimulating others (lysosomes
containing cathepsins B, B + L, and soluble furin to the plasma
membrane). In this respect our results have similarities to the work of
D'Arcy Hart and Young (22) who showed that in mouse macrophages
ammonium chloride blocks phagosome-lysosome fusion but enhances
phagosome-endosome fusion; however, in an earlier study chloroquine was
found to greatly enhance phagosome lysosome fusion (46). A recent
in vitro study of yeast Saccharomyces cerevisiae
by Haas et al. (50) has shown that the homotypic fusion
between vacuoles in yeast is inhibited by bafilomycin A1 arguing that
the presence of a proton gradient seems to be essential for this fusion
event.
Selective Secretion of Hydrolytic Enzymes--
Our results again
highlight the well known ability of macrophages (including macrophage
cell lines) as well as other cells of the immune system to secrete
significant amounts of enzymatically active vacuolar hydrolases, which
has been shown for various hydrolases (51-55) including different
cathepsins (19, 56-58). In a number of these earlier studies the
secretion of hydrolases was significantly enhanced in the presence of
acidotropic drugs, a phenomenon we could confirm here. However, in J774
cells the secretion of hydrolases and an enhancement of this process by
bafilomycin and chloroquine was only seen in the case of cathepsin B, B + L, and soluble furin, whereas cathepsin H and the other six late
endocytic organelle-enriched enzymes were retained by the cell. The
secretion of a subset of lysosomal enzymes could be correlated with a
basal and relatively high level of secretion of internalized HRP from a
late endocytic organelle, probably a lysosome, and again, this
secretion-competent pool could be significantly elevated by acidotropic
drugs. Collectively, these data argue that cathepsin B, B + L, soluble
furin activity, as well as a significant fraction of kinetically late
internalized HRP may be localized to a specialized form of secretory
lysosome.
Although we have no morphological data on the identity of the
hypothetical lysosome/secretory granule, there is an extensive literature describing secretory granules that appear lysosome-like based on their content of marker proteins, as well as lysosomes that
look and behave like secretory granules and are capable of regulated
exocytosis (see Refs. 9 and 59). This phenomenon has been documented in
a wide range of tissues (see Refs. 60-63 and references therein). In
most, if not all of these cases, these two kinds of vesicles appear to
originate from the late endosome rather than the trans-Golgi network
(summarized in Refs. 9 and 59). Moreover, a late endocytic
compartment-derived vesicle has been shown to exocytose bulk membrane
and lumenal contents in antigen presenting cells, a phenomenon believed
to be important for antigen presentation (64). Many earlier studies had
in fact provided EM and biochemical evidence for an exocytosis of
lysosomes in a number of cells, and significantly, this process was
enhanced by chloroquine (65-67). A recent study by Rodriguez et
al. (68) has extended these findings by describing a
calcium-dependent exocytosis of lysosomes in fibroblasts
and epithelial cells. Finally, our data extend the published work of
Rozhin et al. (20), Reddy et al. (57), and
Ulbricht et al. (58) who have all provided evidence for the
regulated secretion of a subset of enzymatically active (mature) acid
hydrolases in different cells, including macrophages.
It has recently been shown that individual vesicles of the late
endocytic organelles can have different pH values (69), and in
conjunction with earlier estimates of lysosomal pH being as low as
4-4.5, it seems that the pH of these vesicles can vary from 4, or even
below, up to neutrality. Our data here, as well as the data of others
(summarized in Ref. 5), argue that late endosomes and lysosomes are
functionally heterogeneous organelles. When one also considers the
dynamic and complex behavior exhibited by endosomes and lysosomes as
seen in video microscopic observations of living
macrophages,4 it becomes
evident that the current textbook notion of a lysosome as a
functionally monogamous, homogenous class of vesicles seems now to be
seriously outdated.
 |
ACKNOWLEDGEMENTS |
We thank Clive Dennison, Edith Elliott
(University of Pietermaritzburg), and Brian Storrie (University of
Virginia) as well as Bernard Hoflack (EMBL) for their critical
comments. The EM immunogold labeling was carried out by Anje
Habermann.
 |
FOOTNOTES |
*
This work was supported by SFB352 of the Deutsche Forschungs
Gemeinschaft and by a grant from the Human Frontier Network (to G. G.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
1
A. Jahraus, T. E. Tjelle, A. Habermann, B. Storrie, O. Ullrich, and G. Griffiths, submitted for publication.
2
The abbreviations used are: HRP, horseradish
peroxidase; BSA, bovine serum albumin; PBS, phosphate-buffered saline;
Z, benzyloxycarbonyl; AMC, 7-amino-4-methylcoumarin; PNS, postnuclear
supernatant.
3
V. Claus, A. Jahraus, T. Tjelle, T. Berg, H. Kirschke, H. Faulstich, and G. Griffiths, unpublished
observations.
4
J. Heuser, personal communication.
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