Portal de Periódicos da CAPES

Cored Tubules are Present in Human Epidermal Langerhans Cells

2003; Elsevier BV; Volume: 120; Issue: 3 Linguagem: Inglês

10.1046/j.1523-1747.2003.12054.x

1523-1747

Dan Lipsker, Umit Ziylan, Danièle Spehner, Fabienne Proamer, Henri de la Salle, Daniel Hanau, Ray McDermott, Jean‐Pierre Cazenave, Bruno Goud, Jean Salamero,

Advancements in Transdermal Drug Delivery

Cored tubules are ultrastructural organelles described to date only in murine cells belonging to the Langerhans cell family and located in the dermis and its draining lymph nodes. These organelles, the function of which is unknown, differ from Birbeck granules and are interestingly not found in murine epidermal Langerhans cells. In this work we demonstrate that cored tubules are present in freshly isolated human epidermal Langerhans cells. The tubules were found to be interconnected with structures known to belong to the early endosomal pathway and could be immunolabeled with gold-conjugated anti-CD1a and anti-Langerin monoclonal antibodies, but only at 37°C. At this temperature such antibodies are able to progress from the early sorting endosomes to the early recycling endosomes, which in human Langerhans cells include the Birbeck granules. These findings strongly suggest that cored tubules form part of the early recycling compartment. Cored tubules are ultrastructural organelles described to date only in murine cells belonging to the Langerhans cell family and located in the dermis and its draining lymph nodes. These organelles, the function of which is unknown, differ from Birbeck granules and are interestingly not found in murine epidermal Langerhans cells. In this work we demonstrate that cored tubules are present in freshly isolated human epidermal Langerhans cells. The tubules were found to be interconnected with structures known to belong to the early endosomal pathway and could be immunolabeled with gold-conjugated anti-CD1a and anti-Langerin monoclonal antibodies, but only at 37°C. At this temperature such antibodies are able to progress from the early sorting endosomes to the early recycling endosomes, which in human Langerhans cells include the Birbeck granules. These findings strongly suggest that cored tubules form part of the early recycling compartment. Langerhans cells are the representatives of the dendritic cell lineage in the epidermis. These cells have the unique ability to capture and process antigens, leave the epidermis, and migrate through the dermis to the afferent lymph nodes, where they present processed antigens to naive T cells and thus initiate specific immunity (Kripke et al., 1990Kripke M.L. Munn C.G. Jeevan A. Tang J.M. Bucana C. Evidence that cutaneous antigen presenting cells migrate to regional lymph nodes during contact sensitisation.J Immunol. 1990; 145: 2833-2838PubMed Google Scholar;Moll et al., 1993Moll H. Fuchs H. Blank C. Rõllinghoff M. Langerhans cells transport Leishmania major from the infected skin to the draining lymph node for presentation to antigen-specific T cells.Eur J Immunol. 1993; 23: 1595-1601Crossref PubMed Scopus (232) Google Scholar). Langerhans cells are the only epidermal cells to constitutively express major histocompatibility complex class II molecules (Klareskog et al., 1977Klareskog L. Malmnas-Tjezrnlund U. Peterson P.A. Epidermal Langerhans cells express Ia antigens.Nature. 1977; 268: 248-250Crossref PubMed Scopus (362) Google Scholar;Rowden et al., 1977Rowden G. Lewis M.G. Sullivan A.K. Ia antigen expression on human epidermal Langerhans cells.Nature. 1977; 268: 247-248Crossref PubMed Scopus (452) Google Scholar), CD1a molecules (Fithian et al., 1981Fithian E. Kung G. Goldstein G. Rubenfeld M. Fenoglio C. Edelson R.L. Reactivity of Langerhans cells with hybridoma antibody.Proc Natl Acad Sci USA. 1981; 78: 2541-2544Crossref PubMed Scopus (439) Google Scholar), and Langerin (Valladeau et al., 2000Valladeau J. Ravel O. Dezutter-Dambuyant C. et al.Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules.Immunity. 2000; 12: 71-81Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar) at their surface. At the ultrastructural level, Langerhans cells also differ from other dendritic cells by the presence of Birbeck granules–distinctive rod-shaped structures of variable length with a central, periodically striated lamella (Birbeck et al., 1961Birbeck M.S. Breathnach A.S. Everall J.D. An electron microscope study of basal melanocytes and high-level clear cells (Langerhans cells) in vitiligo.J Invest Dermatol. 1961; 37: 51-63Abstract Full Text PDF Google Scholar). An additional “specific” ultrastructural organelle called the cored tubule has been reported in mice (Kobayashi and Hoshino, 1978Kobayashi M. Hoshino T. Occurrence of “cored tubule” in the Birbeck granule-containing cells of mice.J Electron Microsc. 1978; 27: 199-205PubMed Google Scholar). It is absent from epidermal Langerhans cells but present in the cytoplasm of cells that are thought to belong to the Langerhans cell family and are found in the dermis and its draining lymph nodes (Kobayashi and Hoshino, 1983Kobayashi M. Hoshino T. Distribution of cored tubule-containing Langerhans cells in the skin and lymph nodes of mice.J Electron Microsc. 1983; 32: 197-206PubMed Google Scholar;Sonoda et al., 1985Sonoda Y. Asano S. Sagami S. Electron microscopic study on Langerhans cells and related cells in lymph nodes of DNCB-sensitive mice.Arch Dermatol Res. 1985; 277: 44-54Crossref PubMed Scopus (16) Google Scholar;Bucana et al., 1992Bucana C.D. Munn C.G. Song M.J. Dunner Jr, K. Kripke M.L. Internalization of Ia molecules into Birbeck granule-like structures in murine dendritic cells.J Invest Dermatol. 1992; 99: 365-373Abstract Full Text PDF PubMed Google Scholar;Bucana et al., 1994Bucana C.D. Tang J.M. Dunner Jr, K. Strickland F.M. Kripke M.L. Phenotypic and ultrastructural properties of antigen-presenting cells involved in contact sensitization of normal and UV-irradiated mice.J Invest Dermatol. 1994; 102: 928-933Crossref PubMed Scopus (24) Google Scholar). A cored tubule displays a rod-shaped profile when sectioned parallel to its long axis, a circular profile when sectioned transversely, and a “core” appearing as a thin line with no periodicity on longitudinal sections or as a dot on cross-sections. These tubules occur either isolated or grouped in the cytoplasm and, when isolated, tend to run in parallel layers and by branching or anastomozing form irregular networks, which often surround spheroid bodies. Sometimes cored tubules seem to be continuous with vacuoles or the rough endoplasmic reticulum but none appear to be connected to the cell membrane. The murine cells “related” to Langerhans cells found in the dermis and its draining lymph nodes contain either cored tubules or Birbeck granules. Intradermal injection of a saline solution, however, induces the appearance of both structures in the same cells, although the Birbeck granules and cored tubules nevertheless remain “scattered independently in the cytoplasm” (Kobayashi and Hoshino, 1983Kobayashi M. Hoshino T. Distribution of cored tubule-containing Langerhans cells in the skin and lymph nodes of mice.J Electron Microsc. 1983; 32: 197-206PubMed Google Scholar). It is noteworthy that, even after saline injection, cored tubules are not observed in epidermal Langerhans cells, whereas in these cells the number of Birbeck granules increases temporarily 10–30 min later (Kobayashi and Hoshino, 1983Kobayashi M. Hoshino T. Distribution of cored tubule-containing Langerhans cells in the skin and lymph nodes of mice.J Electron Microsc. 1983; 32: 197-206PubMed Google Scholar). In this work, we demonstrate for the first time that cored tubules are present in freshly isolated human epidermal Langerhans cells containing Birbeck granules. The relationship of these structures to Birbeck granules, Langerin, and CD1a molecules is discussed and likewise their involvement in the early recycling endosomal compartment of Langerhans cells. DCGM4 (IgG1, recognizing an extracellular epitope of Langerin) (Valladeau et al., 2000Valladeau J. Ravel O. Dezutter-Dambuyant C. et al.Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules.Immunity. 2000; 12: 71-81Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar) and BL6 (IgG1, anti-CD1a) were both provided by Immunotech (Marseille, France). Gold-conjugated Fab fragments of the anti-CD1a monoclonal antibody BL6 (BL6-Au) for electron microscopy were obtained from Aurion (Wageningen, The Netherlands). The anti-Langerin monoclonal antibody DCGM4 was labeled with 10 nm gold particles (Goldsols EM-10, Aurion) (DCGM4-Au) as previously reported (Hanau et al., 1987Hanau D. Fabre M. Schmitt D.A. et al.Human epidermal Langerhans cells internalise by receptor-mediated endocytosis T6 (CD1 “NA1/34) surface antigen: Birbeck granules are involved in the intracellular traffic of the T6 antigen.J Invest Dermatol. 1987; 89: 172-177Abstract Full Text PDF PubMed Google Scholar). Horseradish peroxidase (HRP) type II (P-8250), diaminobenzidine (DAB) (D-5905), and brefeldin A (BFA) (B-7651) were from Sigma Chemical (St. Louis, MO). Cell suspensions were prepared from normal human skin taken from patients undergoing abdominal plastic surgery, as previously described (Hanau et al., 1987Hanau D. Fabre M. Schmitt D.A. et al.Human epidermal Langerhans cells internalise by receptor-mediated endocytosis T6 (CD1 “NA1/34) surface antigen: Birbeck granules are involved in the intracellular traffic of the T6 antigen.J Invest Dermatol. 1987; 89: 172-177Abstract Full Text PDF PubMed Google Scholar). These suspensions initially contained 0.5%–2% freshly isolated Langerhans cells and were enriched in Langerhans cells by gradient centrifugation on Lymphoprep (Flobio, Courbevoie, France), resulting in a Langerhans cell yield of typically 30%–50% of the final cell population. Freshly isolated epidermal cells were either cooled to 19.5°C for 15 min and incubated at 19.5°C for 60 min or warmed to 37°C for 15 min and incubated at 37°C for 25 min with BL6-Au or the gold-conjugated anti-Langerin monoclonal antibody DCGM4 (final dilution 1%), centrifuged at room temperature, and fixed for electron microscopy. Fixation was initiated by adding an equal volume of fixative solution, previously warmed to 37°C, to the cell suspension. The fixative solution contained 3% glutaraldehyde (Electron Microscopy Sciences, Euromedex, Strasbourg, France) and 2% sucrose in 0.1 M sodium cacodylate buffer (both Merck, Darmstadt, Germany) (305 mOsm, pH 7.3). After 5 min the mixture was centrifuged, the supernatant was discarded, and the pellet was resuspended in the same fixative solution and further fixed for 45 min at 37°C. The cells were then washed in 0.1 M sodium cacodylate buffer and postfixed for 1 h at 4°C with 1% osmium tetroxide (Merck) in the same buffer. After further washing in 0.1 M sodium cacodylate buffer, the cells were dehydrated in graded (50%, 70%, 80%, 95%, and 100%) ethanol solutions, incubated overnight in Epon (Electron Microscopy Sciences): absolute alcohol (1:1, vol/vol), and embedded in Epon. Ultrathin sections, stained with lead citrate (Leica, Bron, France) and uranyl acetate (Merck), were examined under a Philips CM 120 BioTwin electron microscope (120 kV). In another series of experiments, freshly isolated epidermal cells were incubated at 37°C for 30 min with BFA (10 μg per ml) and then for 60 min with BFA (10 μg per ml) and HRP (10 mg per ml). The cells were subsequently processed according toTooze and Hollinshead, 1991Tooze J. Hollinshead M. Tubular early endosomal networks in AtT20 and other cells.J Cell Biol. 1991; 115: 635-653Crossref PubMed Scopus (155) Google Scholar andGriffiths et al., 1989Griffiths G. Back R. Marsh M. Quantitative analysis of the endocytic pathway in baby hamster kidney cells.J Cell Biol. 1989; 109: 2703-2720Crossref PubMed Scopus (232) Google Scholar. After washing in phosphate-buffered saline and fixation with 0.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3, for 30 min at room temperature, the cells were washed in cacodylate buffer and incubated with DAB for 1 min. The HRP–DAB reaction was initiated by adding H2O2 to a final concentration of 0.01%. After 30 min in the dark, the reaction was terminated by washing several times in cacodylate buffer and the cells were postfixed in 1% osmium tetroxide, washed again in cacodylate buffer, and further processed as for conventional electron microscopy. Thick and ultrathin sections were examined under a Philips CM 120 BioTwin electron microscope (120 kV), in most cases without poststaining to allow better differentiation of the HRP-DAB reaction product. During electron microscopy studies of the traffic of CD1a and Langerin molecules in freshly isolated human epidermal Langerhans cells (Salamero et al., 2001Salamero J. Bausinger H. Mommaas A.M. et al.CD1a molecules traffic through the early recycling endosomal pathway in human Langerhans cells.J Invest Dermatol. 2001; 116: 401-408Crossref PubMed Scopus (53) Google Scholar), we observed two distinctive types of BL6-Au+ and DCGM4-Au+ rod-shaped organelles, but only when the cells were incubated at 37°C. First, we observed the “classical” Birbeck granules (Fig 1), which appeared as rod-shaped “rigid” structures of variable length with a central, periodically striated lamella. On some sections these structures did not in fact appear rod-shaped but rather disk-shaped, and obliquely or tangentially cut Birbeck granules exhibited a cross-striated pattern or square-lattice appearance, due to the unique arrangement of the particles composing the central lamella. When Birbeck granules were immunogold-labeled with BL6-Au or DCGM4-Au, the gold particles accumulated in their central striated zone, though less with the anti-Langerin antibodies as the level of expression of these molecules is weaker than that of the CD1a molecules at the Langerhans cell surface (data not shown). Second, we observed some much more tortuous rod-shaped structures with an inner central line lacking periodicity, which when sectioned transversely appeared circular with a central dot (Fig 2). These organelles therefore possessed all the ultrastructural characteristics of the “cored tubules” described in mice. Often remaining isolated in the cytoplasm, they were more or less rectilinear and of variable length. These structures could also be continuous, however, with electron-lucent compartments of variable size suggestive of early endosomes, and, by branching or anastomozing, could form pericentriolar networks (Fig 3A, B). Interestingly, when the cored tubules were immunogold-labeled with BL6-Au (Fig 2, Fig 3) or with DCGM4-Au (data not shown), the gold particles were found to be localized on their inner central line. Hence, Birbeck granules and cored tubules can coexist in the cytoplasm of Fi Langerhans cells. Although these two rod-shaped organelles were usually scattered independently, we sometimes observed continuity between the tubular elements of the cored tubules and the characteristic pentalaminar structures of Birbeck granules (Fig 2A).Figure 3Freshly isolated human epidermal Langerhans cells contain networks of cored tubules. Freshly isolated Langerhans cells were warmed to 37°C for 15 min and incubated at 37°C for 25 min with gold-conjugated Fab fragments of the anti-CD1a monoclonal antibody BL6. The branched tubules visible in (A) in the cell center near the centriole (C) display all the morphologic characteristics of “cored tubules”: a thin central line with no periodicity on longitudinal sections (arrows) and a central dot (thin arrowhead) on cross-sectional profiles. Note the presence of (i) a Birbeck granule (arrowhead) radiating from the centriole and (ii) a gold-labeled vacuolar endosome (*) in the center of the cored tubule network. (B) shows a higher magnification of the same network.View Large Image Figure ViewerDownload (PPT) Small G proteins of the Arf (ADP-ribosylation factor) family recruit and assemble protein complexes, leading to the formation of cellular coats. This requires their interaction with Arf exchange factors, which catalyze the exchange of GDP for GTP, a process that can be inhibited by BFA (Donaldson et al., 1992aDonaldson J.G. Finazzi D. Klausner R.D. Brefeldin A inhibits Golgi membrane-catalysed exchange of guanine nucleotide onto ARF protein.Nature. 1992; 360: 350-352Crossref PubMed Scopus (582) Google Scholar;Donaldson et al., 1992bDonaldson J.G. Cassel D. Kahn R.A. Klausner R.D. ADP-ribosylation factor, a small GTP-binding protein, is required for binding of the coatomer protein beta-COP to Golgi membranes.Proc Natl Acad Sci USA. 1992; 89: 6408-6412Crossref PubMed Scopus (376) Google Scholar). Due to its inhibitory effect on coat formation, BFA can induce fusion and redistribution of the membranes of the early endocytic pathway and the trans Golgi network, resulting in the formation of continuous tubular networks (Lippincott-Schwartz et al., 1991Lippincott-Schwartz J. Yuan L. Tipper C. Amherdt M. Orci L. Klausner R.D. Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic.Cell. 1991; 67: 601-616Abstract Full Text PDF PubMed Scopus (667) Google Scholar;Wood et al., 1991Wood S.A. Park J.E. Brown W.J. Brefeldin A causes a microtubule-mediated fusion of the trans-Golgi network and early endosomes.Cell. 1991; 67: 591-600Abstract Full Text PDF PubMed Scopus (285) Google Scholar). In Langerhans cells, where coated structures predominate in the pericentriolar area, BFA promotes interconnections between Birbeck granules and the tubular/vacuolar components of the early endosomal system (McDermott et al., 2002McDermott R. Ziylan U. Spehner D. et al.Birbeck granules are subdomains of the endosomal recycling pathway in human epidermal Langerhans cells which form where Langerin accumulates.Mol Biol Cell. 2002; 13: 317-335Crossref PubMed Scopus (145) Google Scholar). When freshly isolated Langerhans cells were treated at 37°C for 30 min with BFA and then for 60 min with BFA and HRP, we likewise observed interconnections between vacuolar early endosomes and cored tubules (Fig 4). Thus, continuity between cored tubules and elements of the early endosomal pathway seems to occur spontaneously and is enhanced in the presence of BFA. In this work, we show that cored tubules exist at the periphery and in the central and pericentriolar regions of freshly isolated Langerhans cells. Cored tubules are tortuous rod-shaped structures with an inner central line lacking periodicity, which when sectioned transversely appear circular with a central dot. These tubules may be interconnected with structures known to belong to the early endosomal pathway (vesicular/vacuolar early endosomes and Birbeck granules) and can be immunolabeled with the anti-CD1a monoclonal antibody BL6-Au and the anti-Langerin DCGM4-Au, but only at 37°C. The dependence of this labeling of the cored tubules on the temperature conditions bears a resemblance to the traffic of CD1a molecules and Langerin, which permanently recycle between the early endosomal compartments and the cell surface of Langerhans cells in a temperature-dependent manner (Salamero et al., 2001Salamero J. Bausinger H. Mommaas A.M. et al.CD1a molecules traffic through the early recycling endosomal pathway in human Langerhans cells.J Invest Dermatol. 2001; 116: 401-408Crossref PubMed Scopus (53) Google Scholar;McDermott et al., 2002McDermott R. Ziylan U. Spehner D. et al.Birbeck granules are subdomains of the endosomal recycling pathway in human epidermal Langerhans cells which form where Langerin accumulates.Mol Biol Cell. 2002; 13: 317-335Crossref PubMed Scopus (145) Google Scholar). Thus, at 19.5°C two markers of the early endosomal pathway of Langerhans cells, the anti-CD1a monoclonal antibody BL6 and the anti-Langerin monoclonal antibody DCGM4, progress from the cell surface to the EEA1+/Rab5+ early sorting compartments, where they accumulate. Only when the temperature is raised to 37°C do they leave the early sorting compartments and gain access to the Rab11+ early recycling compartments, which in Langerhans cells include the Birbeck granules. Therefore, the fact that cored tubules can be labeled with BL6-Au only when the cells are incubated at 37°C strongly suggests that these structures belong, just like Birbeck granules, to the recycling compartments of Langerhans cells. A relationship between cored tubules and early endosomal compartments is further supported by the findings ofValladeau, 2000Valladeau J. Caractérisation de récepteurs d’endocytose exprimés par les cellules dendritiques humaines et identification de la molécule Langerine. Lyon, 2000: 75-78Google Scholar. This author reported that a point mutation in the proline-rich intracellular motif of Langerin (WPREPPP→WIREPPP) leads to the appearance, in 10%–20% of transfected murine fibroblasts, of authentic networks of cored tubules linked to multivesicular compartments. This proline-rich motif is known to interact with the SH3 domain proteins found in adaptors (Valladeau et al., 2000Valladeau J. Ravel O. Dezutter-Dambuyant C. et al.Langerin, a novel C-type lectin specific to Langerhans cells, is an endocytic receptor that induces the formation of Birbeck granules.Immunity. 2000; 12: 71-81Abstract Full Text Full Text PDF PubMed Scopus (756) Google Scholar). Hence, the appearance of cored tubules, at the expense of tubular recycling endosomes, could be related to interactions between Langerin and adaptors in the recycling compartment. Identification of these adaptors should help to better define the nature of such interactions between Langerin and molecules known to be involved in intracellular sorting. The absence of any connection of cored tubules with the cell membrane would nevertheless suggest that these adaptors should be located only in the cytosol surrounding the recycling compartment. Cored tubules would thus appear to result, like Birbeck granules, from interactions involving a single molecular species, Langerin. These interactions would occur in the recycling compartment where Langerin accumulates. The fact that an intradermal injection of saline solution led to the appearance of both cored tubules and Birbeck granules in murine cells “related” to Langerhans cells and located in the dermis and its draining lymph nodes suggests that activation of these cells could modulate the distribution of Langerin and regulate its traffic, in particular through the recycling compartment. This would explain the observations ofKolde and Knop, 1987Kolde G. Knop J. Different cellular reaction patterns of epidermal Langerhans cells after application of contact sensitizing, toxic, and tolerogenic compounds. A comparative ultrastructural and morphometric time-course analysis.J Invest Dermatol. 1987; 89: 19-23Abstract Full Text PDF PubMed Google Scholar andHanau et al., 1989Hanau D. Fabre M. Schmitt D.A. et al.ATPase and morphological changes in Langerhans cells induced by epicutaneous application of a sensitising dose of DNFB.J Invest Dermatol. 1989; 92: 689-694Abstract Full Text PDF PubMed Google Scholar, who reported that application of a hapten to the skin of, respectively, mice or guinea pigs induced activation of epidermal Langerhans cells and the appearance in their cytoplasm, during the hour following application of the hapten, of numerous Birbeck granules. The authors are especially grateful to J. Mulvihill for excellent editorial assistance and to R. Drillien for critical reading of the manuscript. They would also like to thank J. Leunissen (Aurion, Wageningen, The Netherlands) for preparation and gold-labeling of the Fab fragments of the anti-CD1a monoclonal antibody. D. Lipsker was supported by INSERM and by the Hôpitaux Universitaires de Strasbourg, U. Ziylan by ANRS, and R. McDermott by a grant from ARC. This work was supported by the Etablissement Français du Sang-Alsace, Institut Curie, INSERM, ARMESA, and the Etablissement Français du Sang (CS/2002/018).