Functional Dissociation of the Basolateral Transcytotic Compartment from the Apical Phago-lysosomal Compartment in Human Osteoclasts
School of Anatomy and Human Biology, The University of Western Australia, Crawley, Australia (JM,LF) and Royal Perth Hospital, Perth, Australia (RZ)
Correspondence to: Luis Filgueira, School of Anatomy and Human Biology, The University of Western Australia, MDP: M309, 35 Stirling Highway, Crawley WA 6009, Australia. E-mail: lfilgueira{at}anhb.uwa.edu.au
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Summary |
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(J Histochem Cytochem 53:665670, 2005)
Key Words: osteoclast dendritic cell TRAP Staphylococcus aureus bone osteomyelitis acidic compartment
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Introduction |
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Materials and Methods |
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Surface Markers
Cultured human dendritic cells and osteoclasts were characterized for expression of surface markers for up to 6 weeks in culture using flow cytometry and fluorescence-labeled mouse monoclonal antibodies binding specifically to human CD3, CD8, CD11c, CD14, CD19, CD33, CD80, and MHC class II (BD Biosciences; San Jose, CA).
Phagocytosis
The phagocytic capacity of cultured human dendritic cells and osteoclasts was quantified using flow cytometry and fluorescence confocal microscopy. For that purpose, 2-week cultured cells (105 to 106 cells/ml) were incubated with heat-inactivated fluorescence-labeled (tetramethylrhodamine or AlexaFluor 488) S. aureus (104 to 107 bacteria/ml; Molecular Probes, Eugene, OR) for 496 hr under culture conditions before being fixed with 1% paraformaldehyde in PBS. Cells without bacteria were used as control populations.
Electron Microscopy
Cultured human dendritic cells and osteoclasts were characterized using surface scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The cells were fixed with 2.5% glutaraldehyde in culture medium. For SEM, the cells were processed through critical-point drying and sputtered with 15 nm of gold. They were analyzed with a Philips SEM500 microscope (Philips; Eindhoven, The Netherlands). For TEM, the cells were postfixed with an aqueous solution of 1% OsO4 containing 1.5% K4Fe(CN)6, and embedded in epon. Ultrathin sections were contrasted with lead citrate and uranyl acetate and studied with a Philips CM100 microscope and a JEOL2000 microscope (JEOL USA Inc.; Peabody, MA) (Filgueira et al. 1989).
Confocal Fluorescence Microscopy
The essential part of this study was to investigate whether bacteria-containing endo-lysosomes were acidic and contained TRAP. For that purpose, bacteria-treated (tetramethylrhodamine-labeled) and untreated cells were incubated with 50 µM DAMP-HCl(N-(3-[2,4-dinitrophenyl)amino] propyl)-N-(3-aminopropyl)methylamine; D-1565, Sigma-Aldrich, St Louis, MO) under culture conditions for 1 hr, expecting DAMP to accumulate in acidic vesicles (Megumi et al. 1999). Subsequently, the cells were fixed with 1% paraformaldehyde in PBS. The fixed cells were spun onto glass slides (Star Frost; Knittel Gläser, Braunschweig, Germany) and permeabilized with 0.1% Triton X-100 (ICN Biomedicals; Aurora, OH) in PBS for 1 min. The cells were stained for TRAP activity using a fluorescence-based protocol (Filgueira 2004
). Briefly, the cells were incubated for 15 min with ELF97 substrate (20 µM, E6569; Molecular Probes) in 110 mM acetate buffer (pH 5.2) containing 1.1 mM sodium nitrite and 7.4 mM tartrate (Sigma-Aldrich). The acidic compartment was visualized by using a rabbit anti-dinitrophenol antibody (DakoCytomation; Carpinteria, CA) and a secondary AlexaFluor 488-conjugated goat anti-rabbit IgG antibody (Molecular Probes) (Megumi et al. 1999
). The nuclei were stained with DAPI (4',6-diamidine-2'-phenylindole dihydrochloride, 10 ng/ml; Roche Diagnostics, Mannheim, Germany). After mounting (Dako Fluorescent Mounting Medium; DakoCytomation), the samples were analyzed and documented using confocal microscopy (Bio-Rad MRC 1024, Coherent Enterprise argon ion 250-mW multi-line UV, American Laser 100-mW argon ion multiline laser, Melles-Griot 0.5-mW green helium-neon laser; Bio-Rad, Hercules, CA).
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Results |
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The cells were also processed for SEM and TEM, confirming clear-cut ultrastructural differences between the two cell populations generated from the same common adherent PBMC precursor cells (Figure 1). Dendritic cells were non-adherent or loosely adherent cells of 12 µm to 15 µm in diameter, of varying shapes, with extended membrane protrusions of different shapes. They contained one lobulated nucleus, a large nucleolus, and abundant organelles, including lysosomes. Osteoclasts were large, polarized, adherent cells of 20 µm to 50 µm in diameter and displayed a mushroom-like appearance. They contained one or multiple oval-shaped nuclei with large nucleoli and abundant organelles.
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In dendritic cells, TRAP activity was clearly colocalized with bacteria in endo-lysosomes (Figure 3A). Surprisingly, using the described four-color staining protocol, no overlapping of TRAP-positive, acidic, or bacteria-containing vesicles was detected in osteoclasts (Figures 3B and 3C). These results were repeatedly confirmed using cells from different blood donors and cultured for different time periods (1 to 4 weeks) and different incubation times (496 h). Similar results were also documented when using fluorescence-labeled Escherichia coli (data not shown).
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Discussion |
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First, the cellular human in vitro model was established, and the cells were characterized for ultrastructure, phenotype, and function. Despite having a different aim and knowing that a comparison of mouse osteoclasts and dendritic cell differentiation has already been published (Miyamoto et al. 2001), the present study is the first to compare autologous human monocyte-derived dendritic cells and osteoclasts. As expected, the dendritic cells displayed all the characteristics of immature cells, able to take up and process antigens, including bacteria, and already able to have increased T-cellstimulatory capacity (Filgueira et al. 1996
). In contrast, although derived from the same blood precursor cell population, the osteoclasts showed distinct ultrastructural features different from the dendritic cells, including increased size, multiple nuclei, organized basal adherence (sealing zone and supporting acting ring), and polarization (Figure 1). The phenotype of the monocyte-derived osteoclasts corresponds to the features published by Lader et al. (2001)
, including expression of CD11c. In addition, the osteoclasts were able to resorb calcified extracellular matrix when cultured on dentine slides. In summary, the present study used a well-characterized autologous human in vitro cellular model, comparing human dendritic cells and osteoclasts.
Measurement of bacterial phagocytosis was essential for this study. Immature dendritic cells have already been shown to be potent phagocytes able to take up whole microorganisms, including bacteria (Filgueira et al. 1996; Nagl et al. 2002
). However, little is known about bacterial uptake by human osteoclasts. Therefore, this study also explored the phagocytosis capacity of osteoclasts. For that purpose, the cells were incubated with fluorescence-labeled, heat-inactivated S. aureus and E. coli (data not shown) for up to 96 hr. Bacterial uptake was quantified using fluorescence confocal microscopy and flow cytometry (Figure 2). Both cell populations displayed similar phagocytosis capacity. Being polarized adherent cells, osteoclasts' non-bonerelated uptake is restricted to the apical membrane area. Finally, the present study indicates that human osteoclasts have phagocytosis features similar to those of other cells of the macrophage family.
Processing of bacteria by dendritic cells has been investigated extensively in the past by many research groups (for review see Wick and Ljunggren 1999; Harding et al. 2003
). Dendritic cells have been shown to kill phagocytosed bacteria (Filgueira et al. 1996
; Nagl et al. 2002
). Lysosomal TRAP therefore seems to play an important role in eliminating S. aureus (Räisänen et al. 2001
; Hayman and Cox 2003
). Those published data correlate well with the findings of the present study, which show that there is colocalization of TRAP and bacteria in the phagolysosomes of human dendritic cells. However, this study also indicates that processing of bacteria by osteoclasts differs clearly from what is known for dendritic cells and other macrophages.
Due to their unique function of bone resorption, osteoclasts have distinct, functionally, and ultrastructurally separated vesicular compartments (Figure 4). First, there is one compartment for the supply of enzymes for bone resorption, secreting the vesicular lysosomal content through the basal ruffled border into the resorption lacuna (Väänänen et al. 2000). The acidic vesicles form part of this basal secretory compartment (Megumi et al. 1999
; Hollberg et al. 2002
). Second, there is the vesicular compartment for the transcytosis of resorbed bone material through the cell from the ruffled border to the functional secretory domain (Väänänen et al. 2000
). TRAP itself is located in lysosomal storage granules, which fuse with the transcytotic compartment and help with further degradation of resorbed material (Väänänen et al. 2000
). Third, there is the apical endocytotic compartment, where phagocytosed material, including bacteria, is located, as shown by the present study. In addition, this study indicates that TRAP-containing vesicles do not fuse with the apical phagosomes (Figure 3).
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Most importantly, the physiological separation of apical endocytotic and TRAP-containing transcytotic vesicles may impede the action of TRAP on bacteria and restrain osteoclasts from eliminating endocytosed S. aureus. Finally, the separation of the vesicular compartments in osteoclasts may be responsible for the characteristics of osteomyelitis, where precursor cells of the macrophage family are recruited to the site of inflammation (Kataoka et al. 2000). The bone environment and certain staphylococcal factors direct differentiation of the precursor cells toward osteoclasts with their distinct function of bone resorption (Meghji et al. 1998
), resulting in bone destruction instead of the elimination of bacteria.
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Acknowledgments |
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We thank Miriam Erni, Urs Ziegler, and Peter Groscurth (Institute of Anatomy, University of Zurich, Switzerland), Paul Rigby and Kathryn Heel (Biological Imaging and Analysis Facility, University of Western Australia), and Guy Ben-Ary (Image Acquisition and Analysis Facility, University of Western Australia) for excellent technical support and advice. Electron microscopy was done at the Institute of Anatomy (University of Zurich) and at the Centre for Microscopy and Microanalysis, University of Western Australia.
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Footnotes |
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Literature Cited |
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