The protein tyrosine kinase Hck is located on lysosomal vesicles that are physically and functionally distinct from CD63-positive lysosomes in human macrophages

Catherine Astarie-Dequeker*, Sébastien Carreno, Céline Cougoule and Isabelle Maridonneau-Parini

Institut de Pharmacologie et de Biologie Structurale, Centre National de la Recherche Scientifique UMR 5089, 205 Route de Narbonne, 31077 Toulouse, France

Author for correspondence (e-mail: astarie{at}ipbs)

Accepted September 15, 2001


    SUMMARY
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
In macrophages, lysosomes are suspected to have a heterogenous population of vesicles. This study was thus undertaken to identify and to characterize lysosomal compartments in human macrophages. Hck is a Src-family tyrosine kinase associated with secretory lysosomes in neutrophils and with cytoplasmic vesicles in macrophages that fuse with phagosomes. We identified these Hck-positive vesicles and compared them to CD63-positive, M6PR-negative vesicles known as classical lysosomes. Hck vesicles exhibited lysosomal features. Indeed, Hck-positive vesicles could be loaded with rhodamine-dextran, which has been shown to accumulate in lysosomal compartments. Hck was delivered to zymosan-containing phagosomes at a late stage of the maturation process, which occurs after the fusion with CD63-positive lysosomes. Finally, when mycobacteria were used to prevent phagolysosome biogenesis, Hck was not recruited to phagosomes. Moreover, Hck lysosomes were physically and functionally distinct from CD63-lysosomes. For instance, sucrose induced swelling of CD63-lysosomes without affecting Hck-positive ones. Only CD63-lysosomes fused with phagosomes in a microtubule-dependent manner. Entry of particles through the mannose receptor and Fc{gamma} receptors drove the phagosome towards a fusion with CD63-lysosomes, whereas only Fc{gamma} receptors induced the mobilisation of Hck-lysosomes. This study provides further evidence for the existence of sub-populations of lysosomes in macrophages: one stained by CD63 and another one characterized by the presence of Hck. Therefore, Hck represents a new tool to study the fusion dynamics of lysosomal compartments and their subversion by several intracellular pathogens.

Key words: Hck, Lysosomes, Macrophages


    Introduction
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
One of the crucial steps in the bactericidal responses of macrophages is the fusion of lysosomes with phagosomes. Phagosomes undergo a multi-step maturation process, with sequential fusions with the different compartments of the endocytic pathway until phagolysosomes are formed (Allen and Aderem, 1996; Tjelle et al., 2000). Understanding this fundamental process is crucial, as its subversion is one of the strategies employed by pathogens to survive and replicate inside the host cell.

Lysosomes are usually defined as the kinetically most distal compartment of the endocytic pathway and are devoid of recycling receptors such as mannose 6-phosphate receptor. In macrophages, the situation seems more complex as it is becoming evident that lysosomes encompass distinct endocytic compartments (Claus et al., 1998; Rabinowitz et al., 1992; Tassin et al., 1990). For instance, Rabinowitz et al. could distinguish two kinds of lysosomes exhibiting distinct morphologies. One class has tubular elements and probably corresponds to tubular lysosomes, and the second class has small vesicles (Rabinowitz et al., 1992). Moreover, Claus et al. have demonstrated that lysosomes include two functionally distinct dense compartments, only one of which was found to be secreted in the presence of acidotropic drugs, such as chloroquine or bafilomycine (Claus et al., 1998). Therefore, identification of reliable markers for these lysosomal populations would permit the study of their dynamics along the endocytic/phagocytic pathway and to improve the understanding of the mechanisms involved in inhibition of phagolysosome biogenesis by several pathogens.

Endocytic compartments are usually characterized by the presence of matrix or membrane proteins. In the case of lysosomes, the membrane-associated glycoprotein CD63 is a classic marker. Recently, we demonstrated that the Src-family protein tyrosine kinase Hck exhibits several characteristics of a lysosomal marker in neutrophils. First, we found that Hck is mainly associated with the membrane of azurophil granules, a special class of lysosomes (Möhn et al., 1995; Welch and Maridonneau-Parini, 1997). Second, in cells having engulfed serum-opsonized zymosan, Hck translocates with lysosomes to the phagosomal membrane (Welch and Maridonneau-Parini, 1997). Finally, using mycobacteria to prevent biogenesis of phagolysosomes, we also prevented phagosomal translocation of Hck (N’Diaye et al., 1998). In human macrophages, we have previously demonstrated that Hck is located on vesicles. Although no attempt to identify these vesicles was made, we showed that they were delivered to phagosomes containing zymosan at a maturation step, which is kinetically more distal than the fusion with late endosomes (Astarie-Dequeker et al., 1999).

The present work was undertaken to identify and to characterize Hck-positive vesicles in human macrophages. We investigated whether Hck was associated with a lysosomal compartment in these cells and whether this compartment was distinct from the lysosomal one stained by CD63. Using an immunofluorescence approach, we showed that Hck and CD63 were located on distinct populations of lysosomes that exhibited different functional features.


    Materials and Methods
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Materials
RPMI 1640 with glutamax, Ficoll-Hypaque and penicillin/streptomycin were purchased from Eurobio (les Ulysses, France). Minimum essential medium (MEM), fetal calf serum (FCS) and HEPES were bought from GIBCO (Cergy Pontoise, France). Polystyrene microspheres (1 µm diameter) were purchased from Polysciences Inc. (Warington, PA). Dextran tetramethyl rhodamine lysine fixable (10,000W) was obtained from Molecular Probes (Leiden, Netherlands). Other chemicals were obtained from Sigma (St Louis, MO).

The following antibodies (Abs) were used: rabbit anti-human CI-MPR (cation-independent mannose 6-phosphate receptor) Ab was kindly provided by B. Hoflack (1:500; Institut Pasteur, Lille, France); anti-{alpha} tubulin monoclonal Ab was from M. Wright (1:500; IPBS, Toulouse, France); mouse anti-human CD63 Ab was purchased from CLB (1:100; Amsterdam, Netherlands); rabbit anti-Hck immune serum generated against a peptide corresponding to the N-terminal amino-acid residues 38-52 has been previously characterized (1:100) (Möhn et al., 1995); mouse anti-human Hck Ab was purchased from Transduction Laboratories (1:100; Lexington, KY); rabbit immune serum generated against mycobacteria has been previously characterized (1:50) (Le Cabec et al., 2000). Secondary Abs were purchased from Sigma (St Louis, MO).

Isolation and culture of macrophages
Human peripheral blood monocytes, which were isolated as previously described (Astarie-Dequeker et al., 1999), were cultured on sterile glass coverslips in 24-well tissue culture plates (5x105 cells/well) containing RPMI supplemented with 10% heat-inactivated FCS and antibiotics (100 UI/ml penicillin and 100 µg/ml steptomycin) at 37°C in 5% CO2. The culture medium was renewed the third day, and cells were kept in culture until day six or seven. Before use, macrophages derived from monocytes (MDMs) were washed twice with fresh RPMI and equilibrated for 20 minutes at 37°C in 5% CO2.

Preparation of phagocytic particles
Particles were washed three times in PBS, pH 7.4, counted and routinely added at a ratio of 50 particles per cell. Polystyrene microspheres (1µm in diameter) were coated with 1 mg/ml trimmannoside-BSA (11-12 moles of trimannoside/mole of protein) by non-specific adsorption as previously detailed (Astarie-Dequeker et al., 1999). For experiments requiring IgG-opsonized latex beads, 500 µl of 2.5% bead suspension were washed, then resuspended in PBS containing 13 mg/ml purified human IgG, and incubated for 30 minutes at 37°C to allow adsorption (Koval et al., 1998). The particles were then rinsed and resuspended in PBS at a final concentration of 2x109 particles per ml. Zymosan were opsonized with human serum as previously described (Le Cabec and Maridonneau-Parini, 1994).

The Mycobacterium kansasii (ATCC 124478) was grown and isolated as previously described (N’Diaye et al., 1998). The percentage of viable mycobacteria assessed by serial dilutions and plating on culture medium averaged 85%. Mycobacteria were fluorescently labeled by incubation of 1x109 bacteria with 0.005% FITC in 0.2 M Na2CO3/NaHCO3 and 150 mM NaCl buffer, pH 9.2, for 15 minutes (N’Diaye et al., 1998). Bacteria were then washed twice and resuspended in 1.5 ml PBS, pH 7.4. In some experiments, mycobacteria were serum opsonized (N’Diaye et al., 1998; Peyron et al., 2000). Briefly, FITC-stained bacteria were incubated with a rabbit serum directed against mycobacteria for 25 minutes at 37°C, washed twice and resuspended in PBS, pH 7.4

Labeling of lysosomal compartment with rhodamine-dextran
MDMs were incubated at 37°C with 0.2 mg/ml lysine fixable rhodamine isothiocyanate-conjugated dextran in culture medium for 1 hour, rinced twice with warm medium to remove the non-internalized dextran and incubated for a further 5 hours in dextran-free culture medium. Cells were then fixed for 45 minutes at room temperature with freshly prepared 3.7% paraformaldehyde (PFA), and unreacted aldehyde groups were neutralized with 50 mM NH4Cl for 1 minute. Cells were then washed in PBS, permeabilized for 15 minutes at 37°C with 0.3% Triton-X100 in the presence of 1 mg/ml BSA and processed for immunostaining.

Sucrosome formation
MDMs were incubated for 20 hours in culture medium containing 0.05 M sucrose. After three washes with warm medium and a 5-hour chase in fresh medium, cells were fixed and permeabilized in methanol for 6 minutes at –20°C, washed in PBS containing 0.1% Tween-20 and immunostained.

Phagocytosis and immunofluorescence staining
To enable particle binding, MDMs were incubated at 4°C for 30 minutes with zymosan, latex beads or FITC-mycobacteria. Cells were then extensively washed with cold RPMI and further incubated at 37°C with RPMI to synchronize phagocytosis. At the end of incubation, cells were fixed and permeabilized in methanol. MDMs were finally labeled for 30 minutes at room temperature with Abs directed against markers of interest and revealed by fluorochrome-conjugated secondary Abs (Astarie-Dequeker et al., 1999). For double labeling, cells were first stained with affinity-purified rabbit anti-human Hck Abs, which were revealed by fluorescein-conjugated anti-rabbit IgG Abs, then incubated with mouse Abs directed against endocytic markers and then rhodamine-conjugated anti-mouse Ig Abs. In order to depolymerize microtubules, MDMs were put in contact with zymosan for 20 minutes at 4°C and treated with 10 µM nocodazole or control buffer for a further 10 minutes. Cells were then washed and placed at 37°C for 30 minutes in the presence or absence of the drug.

Fluorescence was visualized using a standard microscope. Phagocytosis was expressed as the percentage of cells having engulfed at least one particle (Astarie-Dequeker et al., 1999). Data are presented as the mean±s.e.m. of the indicated number of experiments performed in duplicate. Where specified, the percentage of phagosomes stained with the marker of interest was determined by counting 100 phagosomes from at least 10 different fields in duplicate samples.

CHO-CR3 transfection and phagocytosis assay
Construction of p61Hck in fusion with GFP has previously been described (Carreno et al., 2000). A point mutation on the myristoylation site (p61G2AHck-GFP) was obtained by inverse PCR of the p61Hck-GFP vector, mutating the glycine 2 codon (GGG) into alanine (GCG). Conformity of mutations was verified by sequencing (Genome Express, Grenoble, France). Human CR3-transfected Chinese Hamster Ovary cells (CHO-CR3) obtained from T. A. Springer (Harvard Medical School, Boston) were cultured and transfected by the DNA/calcium-precipitated method as previously reported (Carreno et al., 2000; Le Cabec et al., 2000).

CHO-CR3 cells transiently transfected with Hck were incubated for 3 hours with serum-opsonized zymosan at a multiplicity of infection of 50:1. Cells were then extensively washed with {alpha}-MEM and fixed with paraformaldehyde. Extracellular particles were revealed as previously described (Le Cabec et al., 2000).


    Results
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
Hck vesicles accumulate fluorescent dextran
In order to verify that Hck vesicles belong to the endocytic pathway, MDMs were allowed to internalize rhodamine-conjugated dextran by fluid-phase endocytosis. After a loading period of 1 hour, followed by a long time-chase of 5 hours in dextran-free medium, Tassin et al. showed that dextran accumulated in lysosomal compartments (Tassin et al., 1990). In human MDMs, fluorescent dextran (Fig. 1B) was found in compartments stained by CD63 (Fig. 1A, merged picture C). Under these conditions, the fluorescent tracer (Fig. 1E) was also detected in vesicles that were positively stained for Hck (Fig. 1D), as shown in the merged picture (Fig. 1F). These results indicate that Hck vesicles belong to the late endocytic pathway.



View larger version (93K):
[in this window]
[in a new window]
 
Fig. 1. Hck vesicles belong to the endocytic pathway. Adherent MDMs were incubated with the endocytic tracer, rhodamine-dextran (Rho-dextran). After a chase of five hours, cells were fixed and labeled with a monoclonal anti-CD63 Ab or polyclonal anti-Hck Abs revealed by FITC-conjugated Abs. Colocalization of Rho-dextran with CD63 (compare B with A) or Hck (compare E with D) appears in yellow in the merged picture (C and F). Data are from one experiment that is representative of four independent experiments. Bar, 7 µm.

 
Hck vesicles fuse with phagosomes after CD63-positive compartments
Fusion of phagosomes with lysosomes is the end point of the phagocytic pathway. To further characterize Hck vesicles, we examined the kinetics of Hck translocation to phagosomes by comparing them with those of CD63. Phagocytosis of zymosan was synchronized as described in the Materials and Methods. Cells were fixed at different time points, double immunolabeling was performed and protein translocation to the phagosomal membrane was visualized as a fluorescent ring surrounding the ingested particle.

To examine whether CD63 was a reliable marker of lysosomes in MDMs, we compared the kinetics of phagosomal recruitment of CD63 and CI-MPR (cation-independent mannose 6-phosphate receptor), a late endosomal protein not detected in lysosomes (Geuze et al., 1988; Griffiths et al., 1988; Griffiths et al., 1990). Five minutes after initiation of phagocytosis, CI-MPR was present on 100% of phagosomes (see arrows Fig. 2Aa), but it totally disappeared five minutes later (see arrows Fig. 2Ac). By comparison, CD63 was undetectable on phagosomes at five minutes (see arrows on Fig. 2Ab) but was present on 38% of phagosomes at 10 minutes (see arrows Fig. 2Ad) and on 60% at 30 minutes (Fig. 2Af). From these data showing that CD63 is delivered to phagosomes after a late endosomal marker is, we concluded that CD63 is mainly associated with a lysosomal compartment in MDMs.



View larger version (96K):
[in this window]
[in a new window]
 
Fig. 2. Chronological acquisition of CI-MPR, CD63 and Hck during the maturation of phagosomes containing zymosan. MDMs were infected with zymosan under synchronized conditions. At different post-infection times (5 (a,b), 10 (c,d) or 30 (e,f) minutes), cells were fixed and permeabilized in methanol. Double immunostaining was performed using Abs directed against CD63 and CI-MPR or Hck. We focused on phagosomes, and the presence of the markers at the phagosomal membrane was determined by a fluorescent ring (see arrows). (A) CI-MPR, a marker of late endosomes (a,c,e) was delivered to phagosomes before CD63 (b,d,f) (arrows). (B) CD63 (a,c,e) was delivered to phagosomes before Hck (b,d,f) (arrows). Occasionally Hck and CD63 were found on the same phagosome (arrowhead). Data are from one experiment that is representative of two to four independent experiments. Bar, 7 µm.

 
Double immunolabeling of CD63 and Hck was then carried out. Five minutes after initiation of phagocytosis, phagosomes were negative for both CD63 and Hck (Fig. 2Ba,b, see arrows). Five minutes later, half of them acquired CD63 (Fig. 2Bc, see arrows) but were negative for Hck (Fig. 2Bd, see arrows). At 30 minutes, Hck was detectable on 80% of phagosomes (Fig. 2Bf. see arrows), some of which were occasionally CD63 positive (Fig. 2Be,f, see arrowheads). These experiments provide evidence that Hck translocates onto phagosomes at a late stage of the maturation process of phagosomes, after mobilization of CD63-positive lysosomes. Such kinetics, combined with the ability of Hck-vesicles to accumulate dextran after a long period of chase, suggested that Hck-vesicles exhibit lysosomal features.

We have previously shown that p61Hck is the isoform that is mainly associated with lysosomes (Möhn et al., 1995; Welch and Maridonneau-Parini, 1997). It is also associated with the Golgi apparatus in transfected cell lineages (Carreno et al., 2000) but not in neutrophils (Möhn et al., 1995) nor in human MDMs (data not shown). A small fraction of p61Hck is also present in the cytosol of neutrophils (Möhn et al., 1995; Welch and Maridonneau-Parini, 1997). However, the presence of Hck on phagosomes is not due to a translocation from the cytosol as this fraction of p61Hck remained constant during phagocytosis of zymosan by human neutrophils although its lysosomal fraction decreased (Welch and Maridonneau-Parini, 1997). Similar experiments performed in human MDMs confirmed that the level of p61Hck in the cytosol, as analysed by western blot, did not vary during phagocytosis (data not shown). An additional approach was used that consisted of transfection of p61Hck in CHO cells stably expressing the human Complement receptor 3, CHO-CR3 cells. CHO cells are non-phagocytic cells that have the capacity to ingest complement-opsonized particles when they express CR3 (Le Cabec et al., 2000). When these cells are transfected with the p61 Hck isoform, lysosomes are Hck positive (Carreno et al., 2000). Under these conditions, opsonized zymosan accumulated in phagosomes that were also positive for Hck (Fig. 3A). In contrast, the non-myristoylated variant of p61Hck, which is unable to associate with lysosomes and remains in the cytosol (Carreno et al., 2000), did not translocate to phagosomes (Fig. 3B). Taken together, these results suggest that the localization of Hck on phagosomes is not due to a translocation from the cytosol but rather due to a translocation from lysosomal vesicles.



View larger version (53K):
[in this window]
[in a new window]
 
Fig. 3. Phagosomal translocation of p61Hck lysosomal isoform in transfected CHO-CR3. CHO-CR3 cells transiently expressing the lysosomal p61Hck-GFP (A) or cytosolic p61G2AHck-GFP (B) were incubated with serum-opsonized zymosan. Cells were fixed and non-permeabilized, extracellular zymosan particles were seen with primary Abs revealed by secondary TRITC-conjugated Abs. Translocation of p61Hck-GFP on phagosomes is visualized by a green fluorescent ring (A, arrow) although no fluorescent ring was seen on the phagosomal membrane (black hole) in cells expressing the cytosolic form of Hck (B, arrowhead) as observed by confocal microscopy. Bar, 7 µm.

 
Hck vesicles are physically distinct from CD63-positive lysosomes
Our next experiment was to determine if Hck and CD63 are located on distinct compartments. Sucrose has been previously described to induce the osmotic swelling of lysosomal compartments (DeCourcy and Storrie, 1991; Jahraus et al., 1994; Montgomery et al., 1991). This prompted us to test the influence of sucrose on Hck- and CD63-positive compartments. As represented in Fig. 3, MDMs pulsed with 0.05 M sucrose for 20 hours and chased for five hours exhibited large vacuoles. In agreement with previous observations (DeCourcy and Storrie, 1991; Jahraus et al., 1994; Montgomery et al., 1991), these vacuoles were CI-MPR-free (Fig. 4A) and CD63 positive (Fig. 4B,D). Interestingly, these structures were devoid of Hck staining (Fig. 4C), even when the chase time was increased to 24 hours (data not shown). Thus, sucrose allowed us to distinguish Hck lysosomes from CD63 lysosomes, supporting our previous report that CD63 and Hck do not colocalize in resting MDMs (Astarie-Dequeker et al., 1999). This led us to propose that lysosomes encompass at least two subpopulations characterized by either CD63 or Hck.



View larger version (100K):
[in this window]
[in a new window]
 
Fig. 4. Sucrose induces osmotic swelling of CD63-positive lysosomes without affecting Hck vesicles. Sucrosomes were formed by incubating MDMs in culture medium supplemented with 0.05 M sucrose for 20 hours. After a chase of five hours in sucrose-free culture medium, cells were fixed and permeabilized in methanol. Double immunolabeling was performed using Abs directed against CD63 and CI-MPR or Hck. Sucrose induced the formation of large vacuoles surrounded by CD63 (B,D) but devoid of CI-MPR (A) and Hck (C) (arrows). For A and C, sucrosomes are seen as black holes surrounded by cytoplasm without specific immunolabeling. Data are from one experiment that is representative of three independent experiments. Bar, 7 µm.

 
Role of microtubules in the mobilisation of Hck and CD63 lysosomes
We examined the functional characteristics of the two lysosomal compartments. First, we took advantage of previous data showing that microtubules are implicated in the intracellular movements of lysosomes (Blocker et al., 1998; Gruenberg et al., 1989) and in the transfer of late endocytic markers to phagosomes (Blocker et al., 1996; Desjardins et al., 1994; Funato et al., 1997). We therefore studied whether the delivery of Hck and CD63 to zymosan-containing phagosomes was dependent on the integrity of the microtubule network by observing the effect of nocodazole, a microtubule-depolymerizing agent. We checked that cell treatment with 10 µM nocodazole efficiently depolymerised microtubules in resting macrophages (data not shown) and did not affect the percentage of cells with internalized zymosan (28±4 and 25±1% in the absence and the presence of the drug, n=2). In the presence of nocodazole, the percentage of CD63-positive phagosomes was decreased two-fold compared to untreated cells, whereas the number of Hck-positive phagosomes remained constant (Fig. 5). This set of experiments indicates that although CD63 lysosomes require, at least in part, the integrity of the microtubule network, Hck lysosomes fuse equally well with phagosomes in the presence or absence of nocodazole.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. Effects of nocodazole on the fusion of Hck and CD63 lysosomes with phagosomes containing zymosan. MDMs in contact with zymosan for 20 minutes at 4°C were further incubated for 10 minutes with nocadazole (10 µM) or control buffer. Phagocytosis was initiated by transferring the cells to 37°C for 30 minutes in the absence or in the presence of nocodazole. Double immunolabeling was then performed with anti-CD63 and anti-Hck Abs. The percentage of positive phagosomes for each marker in the absence (white bar) or in the presence of nocodazole (black bar) was determined by counting 100 phagosomes from at least 10 different fields in duplicate samples. Data are means± s.d. of three experiments.

 
Role of phagocytic receptors in the mobilisation of Hck and CD63 lysosomes
Since receptors involved in the ingestion of a particle can influence the biogenesis of phagolysosomes (Armstrong and Hart, 1975; Bouvier et al., 1994; Joiner et al., 1990), we examined whether the fusion of Hck and CD63 lysosomes with phagosomes could be controlled by phagocytic receptors. To address this question, MDMs were incubated with latex beads coated with trimannoside-BSA or IgG to allow their internalization through the mannose receptor (MR) (Astarie-Dequeker et al., 1999) or Fc{gamma} receptors (Fc{gamma}Rs), respectively. We have previously reported that phagocytosis through MR allows fusion of phagosomes with Lamp-1 vesicles but not with Hck vesicles (Astarie-Dequeker et al., 1999), and previous work has demonstrated that Fc{gamma}Rs are coupled to the biogenesis of phagolysosomes (Armstrong and Hart, 1975; Bouvier et al., 1994). Experiments with synchronized phagocytosis followed by a chase of two hours were performed. As shown in Fig. 6A, phagosomes containing mannosylated beads remained free of Hck (see arrows) even when the time of phagosome maturation was increased up to three hours (data not shown). In contrast, phagosomes were positive for CD63 (Fig. 6B, see arrows).



View larger version (63K):
[in this window]
[in a new window]
 
Fig. 6. Role of phagocytic receptors in the fusion of phagosomes with Hck and CD63 lysosomes. MDMs were incubated with latex beads coated with mannosylated-BSA (Man-BSA-latex) or human IgG (IgG-latex), and phagocytosis was performed under synchronized conditions. Two hours post-infection, cells were fixed and permeabilized with methanol, and double immunostaining was performed using Abs directed against CD63 or Hck. Mannosylated-latex beads resided in phagosomes negative for Hck (A) and positive for CD63 (B) (arrows). IgG-coated latex beads resided in phagosomes positive for Hck (C) and CD63 (D) (arrows). Bar, 8 µm. (E) Recruitment of Hck and CD63 to phagosomes containing IgG-latex beads was quantified as a percentage of the phagosomes positive for each marker at different time points. Data are from one experiment that is representative of four independent experiments.

 
When latex beads entered the cells through Fc{gamma}Rs, both Hck and CD63 were recruited on phagosomes (Fig. 6C,D). However, their kinetics of recruitment were different, as depicted in Fig. 6E. Although the percentage of CD63-positive phagosomes progressively increased as phagosomes matured, reaching a plateau at 30 minutes, all phagosomes were Hck positive a few minutes after particle ingestion. Then, Hck staining rapidly decreased and disappeared at 45 minutes. A second wave of recruitment occurred at late stages of phagosome maturation, with approximately 80% of phagosomes positive for Hck at 90 minutes (Fig. 6E). The very rapid and transient association of Hck with nascent phagosomes was not compatible with our current knowledge of the biogenesis of phagolysosomes in macrophages (Tjelle et al., 2000). It probably reflects the association and clustering of Hck with Fc{gamma}Rs at the plasma membrane as previously reported (Ghazizadeh et al., 1994; Wang et al., 1994). Although the amount of Hck at the plasma membrane is under the detection limits of fluorescence microscopy in resting macrophages and neutrophils (Astarie-Dequeker et al., 1999; Welch et al., 1996), it is easy detected in transiently transfected Hela cells (Carreno et al., 2000). The second wave of Hck translocation occurring after the fusion of CD63 lysosomes probably reflects the fusion of Hck lysosomes with phagosomes. Therefore, both MR and Fc{gamma}Rs are capable of inducing the mobilisation of CD63-positive lysosomes although only particle entry through Fc{gamma}Rs leads to the fusion of phagosomes with Hck-positive lysosomes. This further emphasizes the functional differences between these two lysosomal compartments.

Behaviour of Hck lysosomes in MDMs infected with mycobacteria
Pathogenic mycobacteria reside in phagosomes that display a marked reluctance to fuse with lysosomes (Armstrong and Hart, 1975; Clemens and Horwitz, 1995; Crowle et al., 1991; Ullrich et al., 1999; Via et al., 1997), whereas the fusion is restored when bacteria are coated with antibodies (Armstrong and Hart, 1975). This prompted us to look at the mobilization of CD63 and Hck lysosomes in cells infected with slow-growing mycobacteria, M. kansasii. Synchronized phagocytosis of mycobacteria was performed. Two hours post-infection, we noticed that phagosomes containing either non-opsonized (Fig. 7A, see arrows) or immune-serum-opsonized M. kansasii (Fig. 7C, see arrows) were surrounded by a CD63 ring (Fig. 7B,D, see arrows). This corroborates previous data showing that phagosomes containing pathogenic mycobacteria are surprisingly accessible to proteins of late endocytic compartments, such as Lamp-1 and to cathepsin D (Ullrich et al., 1999; Xu et al., 1994). However, these proteins are also present in post-Golgi vesicles and thereby gain access to mycobacteria-harboring phagosomes arrested at an early endosomal/recycling step (Ullrich et al., 1999).



View larger version (77K):
[in this window]
[in a new window]
 
Fig. 7. Hck lysosomes fuse with phagosomes containing M. kansasii only when mycobacteria are serum-opsonized. MDMs were infected with non-opsonized or immune-serum-opsonized FITC-M. kansasii under synchronized conditions. Two hours post-infection, cells were fixed and immunolabeled for CD63 or Hck. Secondary Abs were TRITC-conjugated. We focused on phagosomes. Phagosomes containing non-opsonized or serum-opsonized mycobacteria (A,C) were positive for CD63 (B,D) (arrows). Some non-opsonized mycobacteria (A) were also found in CD63-negative phagosomes (B) (arrowheads). Phagosomes containing non-opsonized mycobacteria (E) were negative for Hck (F) (arrows) although phagosomes harboring serum-opsonized mycobateria (G) were positive for Hck (H) (arrows). Data are from one experiment that is representative of three independent experiments. Bar, 5.5 µm.

 
When internalized through a non-opsonic pathway, M. kansasii were found in phagosomes (Fig. 7E, arrows) devoid of Hck staining (Fig. 7F, arrows). Similar observations were made for longer infection times, up to 24 hours (data not shown). Interestingly, when mycobacteria were opsonized with immune serum, phagosomes containing M. kansasii (Fig. 7G, arrows) exhibited a strong Hck labeling (Fig. 7H, arrows), indicating that entry of mycobacteria through opsonic receptors renders phagosomes fusogenic with Hck lysosomes.


    Discussion
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 
In this study, we show that lysosomes of human macrophages have at least two separate compartments with distinct functionality. A first class of structures was stained by the classic lysosomal marker CD63 and a second class was characterized by the presence of the Src-family protein tyrosine kinase, Hck.

During the maturation process of phagosomes in macrophages, Hck located on vesicular structures is recruited to the phagosomal membrane, following recruitment of the late endosomal protein, Lamp-1 (Astarie-Dequeker et al., 1999). We now demonstrate that Hck vesicles exhibit lysosomal characteristics. They belong to the endocytic pathway, as they accumulate the fluid-phase endocytic marker dextran under experimental conditions that drive the tracer in lysosomes (Tassin et al., 1990) as confirmed herein by colocalization of rhodamine-dextran with CD63-positive lysosomes. This is consistent with our recent findings in Hela cells transiently expressing Hck, showing that dextran reaches Hck-positive vesicles (Carreno et al., 2000). We also show that the fusion of Hck vesicles with phagosomes occurs at a very late stage of phagosome maturation, in agreement with lysosomal behaviour. Indeed, zymosan particles reside in phagosomes that successively interact with late endosomes (CI-MPR positive) then with classic lysosomes (CI-MPR negative and CD63 positive) and finally with Hck vesicles. We demonstrate that the behaviour of Hck vesicles strictly correlates with the previously reported behaviour of lysosomes in macrophages infected with mycobacteria (Armstrong and Hart, 1975). For instance, when macrophages are infected with pathogenic mycobacteria, the fusion between lysosomes and phagosomes is prevented (Armstrong and Hart, 1975), and the fusion with Hck vesicles did not take place (this report). Conversely, opsonization of mycobacteria with immune serum restores the fusion of their phagosomes with lysosomes (Armstrong and Hart, 1975) and also the fusion with Hck vesicles (this report). Thus, Hck vesicles and lysosomes share common properties.

It is evident that lysosomes are not a homogeneous class of organelles. In macrophages, at least, two classes of lysosomes have been proposed (Claus et al., 1998; Rabinowitz et al., 1992; Tassin et al., 1990). This report further supports this notion as we have been able to characterize a lysosomal compartment that is physically and functionally distinct from CD63 lysosomes. First, the osmotically active solute sucrose accumulates in CD63 lysosomes but not in Hck vesicles. Second, CD63 and Hck are sequentially recruited along the maturation pathway of phagosomes. Third, although a microtubule-depolymerizing drug has no effect on the fusion of Hck lysosomes with phagosomes containing zymosan, it strongly affects the fusion of CD63 lysosomes. Previous data provide evidence that fusion of phagosomes with late organelles of the endocytic pathway is dependent on the integrity of microtubules (Blocker et al., 1996; Desjardins et al., 1994; Funato et al., 1997). Other reports show that microtubules are not rate limiting for either phagosome-lysosome fusion or for degradation of the phagocytosed content (Knapp and Swanson, 1990; Pesanti and Axline, 1975a; Pesanti and Axline, 1975b). These apparent conflicting results could be reconciled if lysosomes are considered as heterogeneous organelles. Our data also indicate that fusion of Hck lysosomes with phagosomes does not require previous mobilization of CD63 lysosomes. Finally, we show that the receptors involved in phagocytosis are crucial in triggering the fusion process between phagosomes and Hck-positive lysosomes. When latex beads were internalized through Fc{gamma}R, Hck vesicles fused with phagosomes, although they did not when beads were internalized through the mannose receptor. These results are in agreement with other reports showing that IgG opsonisation of particles triggers the fusion of lysosomes with phagosomes (Bouvier et al., 1994; Joiner et al., 1990). In contrast, the fusion of CD63 lysosomes with phagosomes takes place independently of the receptor of entry. We conclude that Hck belongs to a subpopulation of lysosomes that is mobilized under a receptor-regulated microtubule-independent manner.

Expression of Hck is mainly restricted to phagocytes that play a critical role in the killing of ingested microorganisms. Neutrophils and monocytes are characterized by the presence of ‘specialized lysosomes’ that contain bactericidal proteins in addition to the ubiquitous lysosomal enzymes. Exocytosis of the specialized lysosomal content occurs in a regulated manner, and we have shown that it correlates with the activation of Hck associated with lysosomes (N’Diaye et al., 1998; Welch and Maridonneau-Parini, 1997). During the differentiation process of monocytes into macrophages, most of the bactericidal enzymes disappear from their lysosomes (Van Furth, 1992), and one could question whether they also lose their regulated secretion machinery. However, a subpopulation of regulated secretory lysosomes has been suspected in macrophages (Claus et al., 1998), and we have previously shown that fusion of Hck-positive vesicles with phagosomes correlates with activation of the kinase activity (Astarie-Dequeker et al., 1999). In addition, we show herein that fusion of Hck-positive, CD63-negative lysosomes is regulated by the route of entry of particles, as expected for a regulated exocytic compartment. Therefore we propose that, in macrophages, Hck is a marker of a regulated secretory CD63-negative lysosomal compartment. This is supported by our recent finding that when Hck is transiently expressed in human epithelial HeLa cells, which are not expected to contain a population of secretory lysosomes, the kinase colocalized with CD63 (Carreno et al., 2000). In conclusion, Hck is associated with a subpopulation of lysosomes in macrophages that present several criteria of a regulated secretory compartment. We propose that Hck represents a useful tool to investigate the fusion dynamics of this lysosomal compartment with phagosomes and its regulation by pathogens.


    ACKNOWLEDGMENTS
 
We thank Dr A. Labrousse for critical reading of the manuscript. This work was supported by Sidaction and ARC Grand Sud n° 7542


    REFERENCES
 Top
 SUMMARY
 Introduction
 Materials and Methods
 Results
 Discussion
 REFERENCES
 

Allen, L. A. and Aderem, A. (1996). Mechanisms of phagocytosis. Curr. Opin. Immunol. 8, 36-40.[Medline]

Armstrong, J. A. and Hart, P. D. (1975). Phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. Reversal of the usual nonfusion pattern and observations on bacterial survival. J. Exp. Med. 142, 1-16.[Abstract]

Astarie-Dequeker, C., N’Diaye, E. N., LeCabec, V., Rittig, M., Prandi, J. and Maridonneau-Parini, I. (1999). The mannose receptor mediates uptake of pathogenic and nonpathogenic mycobacteria and bypasses bactericidal responses in human macrophages. Infect. Immun. 67, 469-477.[Abstract/Free Full Text]

Blocker, A., Severin, F. F., Habermann, A., Hyman, A. A., Griffiths, G. and Burkhardt, J. K. (1996). Microtubule-associated protein-dependent binding of phagosomes to microtubules. J. Biol. Chem. 271, 3803-3811.[Abstract/Free Full Text]

Blocker, A., Griffiths, G., Olivo, J. C., Hyman, A. A. and Severin, F. F. (1998). A role for microtubule dynamics in phagosome movement. J. Cell Sci. 111, 303-311.[Abstract/Free Full Text]

Bouvier, G., Benoliel, A. M., Foa, C. and Bongrand, P. (1994). Relationship between phagosome acidification, phagosome-lysosome fusion, and mechanisms of particle ingestion. J. Leuk. Biol. 55, 729-734.[Abstract]

Carreno, S., Gouze, M. E., Schaak, S., Emorine, L. J. and Maridonneau-Parini, I. (2000). Lack of palmitoylation redirects p59Hck from the plasma membrane to p61Hck positive lysosomes. J. Biol. Chem. 275, 36223-36229.[Abstract/Free Full Text]

Claus, V., Jahraus, A., Tjelle, T., Berg, T., Kirschke, H., Faulstich, H. and Griffiths, G. (1998). Lysosomal enzyme trafficking between phagosomes, endosomes, and lysosomes in J774 macrophages. Enrichment of cathepsin H in early endosomes. J. Biol. Chem. 273, 9842-9851.[Abstract/Free Full Text]

Clemens, D. L. and Horwitz, M. A. (1995). Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J. Exp. Med. 181, 257-270.[Abstract]

Crowle, A. J., Dahl, R., Ross, E. and May, M. H. (1991). Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic. Infect. Immun. 59, 1823-1831.[Medline]

DeCourcy, K. and Storrie, B. (1991). Osmotic swelling of endocytic compartments induced by internalized sucrose is restricted to mature lysosomes in cultured mammalian cells. Exp. Cell Res. 192, 52-60.[Medline]

Desjardins, M., Huber, L. A., Parton, R. G. and Griffiths, G. (1994). Biogenesis of phagolysosomes proceeds through a sequential series of interactions with the endocytic apparatus. J. Cell Biol. 124, 677-688.[Abstract]

Funato, K., Beron, W., Yang, C. Z., Mukhopadhyay, A. and Stahl, P. D. (1997). Reconstitution of phagosome-lysosome fusion in streptolysin O-permeabilized cells. J. Biol. Chem. 272, 16147-16151.[Abstract/Free Full Text]

Geuze, H. J., Stoorvogel, W., Strous, G. J., Slot, J. W., Bleekemolen, J. E. and Mellman, I. (1988). Sorting of mannose 6-phosphate receptors and lysosomal membrane proteins in endocytic vesicles. J. Cell Biol. 107, 2491-2501.[Abstract]

Ghazizadeh, S., Bolen, J. B. and Fleit, H. B. (1994). Physical and functional association of Src-related protein tyrosine kinases with Fc gamma RII in monocytic THP-1 cells. J. Biol. Chem. 269, 8878-8884.[Abstract/Free Full Text]

Griffiths, G., Hoflack, B., Simons, K., Mellman, I. and Kornfeld, S. (1988). The mannose 6-phosphapte receptor and the biogenesis of lysosomes. Cell 52, 329-341.[Medline]

Griffiths, G., Matteoni, R., Bach, R. and Hoflack, B. (1990). Characterization of the cation-independent mannose 6-phosphate receptor- enriched prelysosomal compartment in NRK cells. J. Cell Sci. 95, 441-461.[Abstract]

Gruenberg, J., Griffiths, G. and Howell, K. E. (1989). Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J. Cell Biol. 108, 1301-1316.[Abstract]

Jahraus, A., Storrie, B., Griffith, G. and Dejardins, M. (1994). Evidence for retrograde traffic between terminal lysosomes and the prelysosomal/late endosome compartment. J. Cell Sci. 107, 145-157.[Abstract/Free Full Text]

Joiner, K. A., Fuhrman, S. A., Miettinen, H. M., Kasper, L. H. and Mellman, I. (1990). Toxoplasma gondii: fusion competence of parasitophorous vacuoles in Fc receptor-transfected fibroblasts. Science 249, 641-646.[Medline]

Knapp, P. E. and Swanson, J. A. (1990). Plasticity of the tubular lysosomal compartment in macrophages. J. Cell Sci. 95, 433-439.[Abstract]

Koval, M., Preiter, K., Adles, C., Stahl, P. D. and Steinberg, T. H. (1998). Size of IgG-opsonized particles determines macrophage responses during internalization. Exp. Cell Res. 242, 265-273.[Medline]

Le Cabec, V. and Maridonneau-Parini, I. (1994). Annexin 3 is associated with cytoplasmic granules in neutrophils and monocytes and translocates to the plasma membrane in activated cells. Biochem. J. 303, 481-487.[Medline]

Le Cabec, V., Cols, C. and Maridonneau-Parini, I. (2000). Nonopsonic phagocytosis of zymosan and Mycobacterium kansasii by CR3 (CD11b/CD18) involves distinct molecular determinants and is or is not coupled with NADPH oxidase activation. Infect. Immun. 68, 4736-4745.[Abstract/Free Full Text]

Möhn, H., Cabec, V. L., Fisher, S. and Maridonneau-Parini, I. (1995). The src-family protein tyrosine kinase p59 hck is located on the secretory granules in human neutrophils and translocates toward the phagosomes during cell activation. Biochem. J. 309, 657-665.[Medline]

Montgomery, R. R., Webster, P. and Mellman, I. (1991). Accumulation of indigestible substances reduces fusion competence of macrophage lysosomes. J. Immunol. 147, 3087-3095.[Abstract/Free Full Text]

N’Diaye, E. N., Darzacq, X., Astarie-Dequeker, C., Daffé, M., Calafat, J. and Maridonneau-Parini, I. (1998). Fusion of azurophil granules with phagosomes and activation of the tyrosine kinase Hck are specifically inhibited during phagocytosis of mycobacteria by human neutrophils. J. Immunol. 161, 4983-4991.[Abstract/Free Full Text]

Pesanti, E. L. and Axline, S. G. (1975a). Colchicine effects on lysosomal enzyme induction and intracellular degradation in the cultivated macrophage. J. Exp. Med. 141, 1030-1046.[Abstract]

Pesanti, E. L. and Axline, S. G. (1975b). Phagolysosome formation in normal and colchicine-treated macrophages. J. Exp. Med. 142, 903-913.[Abstract]

Peyron, P., Bordier, C., N’Diaye, E. N. and Maridonneau-Parini, I. (2000). Nonopsonic phagocytosis of Mycobacterium kansasii by human neutrophils depends on cholesterol and is mediated by CR3 associated with glycosylphosphatidylinositol-anchored proteins. J. Immunol. 165, 5186-5191.[Abstract/Free Full Text]

Rabinowitz, S., Horstmann, H., Gordon, S. and Griffiths, G. (1992). Immunocytochemical characterization of the endocytic and phagolysosomal compartments in peritoneal macrophages. J. Cell Biol. 116, 95-112.[Abstract]

Tassin, M., Lang, T., Antoine, J. C., Hellio, R. and Ryter, A. (1990). Modified lysosomal compartment as carrier of slowly and non-degradable tracers in macrophages. Eur. J. Cell Biol. 52, 219-228.[Medline]

Tjelle, T. E., Lovdal, T. and Berg, T. (2000). Phagosome dynamics and function. Bioessays 22, 255-263.[Medline]

Ullrich, H. J., Beatty, W. L. and Russell, D. G. (1999). Direct delivery of procathepsin D to phagosomes: implications for phagosome biogenesis and parasitism by Mycobacterium. Eur. J. Cell Biol. 78, 739-748.[Medline]

Van Furth, R. (1992). Development and distribution of mononuclear phagocytes. In inflammation: Basic principles and clinical correlates (ed. J. Galli, Goldstein, IM and Snyderman, R), pp. 325-351. New York: Raven Press.

Via, L. E., Deretic, D., Ulmer, R. J., Hibler, N. S., Huber, L. A. and Deretic, V. (1997). Arrest of mycobacterial phagosome maturation is caused by a block in vesicle fusion between stages controlled by rab5 and rab7. J. Biol. Chem. 272, 13326-13331.[Abstract/Free Full Text]

Wang, A. V., Scholl, P. R. and Geha, R. S. (1994). Physical and functional association of the high affinity immunoglobulin G receptor (Fc gamma RI) with the kinases Hck and Lyn. J. Exp. Med. 180, 1165-1170.[Abstract]

Welch, H. and Maridonneau-Parini, I. (1997). Hck is activated by opsonized Zymosan and A23187 in distinct subcellular fractions in human granulocytes. J. Biol. Chem. 272, 102-109.[Abstract/Free Full Text]

Welch, H., Mauran, C. and Maridonneau-Parini, I. (1996). Non-receptor protein tyrosine kinases in neutrophil activation. Methods Companion Methods in Enzymol. 9, 607-618.

Xu, S., Cooper, A., Sturgill-Koszycki, S., van Heyningen, T., Chatterjee, D., Orme, I., Allen, P. and Russell, D. G. (1994). Intracellular trafficking in Mycobacterium tuberculosis- and Mycobacterium avium-infected macrophages. J. Immunol. 153, 2568-2578.[Abstract/Free Full Text]