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
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SUMMARY |
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Key words: Hck, Lysosomes, Macrophages
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Introduction |
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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 (NDiaye 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.
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Materials and Methods |
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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- 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 (NDiaye 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 (NDiaye 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 (NDiaye 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 -MEM and fixed with paraformaldehyde. Extracellular particles were revealed as previously described (Le Cabec et al., 2000).
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Results |
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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.
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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.
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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).
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Discussion |
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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 FcR, 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 (NDiaye 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.
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ACKNOWLEDGMENTS |
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