CD208/Dendritic Cell-Lysosomal Associated Membrane Protein Is a Marker of Normal and Transformed Type II Pneumocytes
2004; Elsevier BV; Volume: 164; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)63174-4
ISSN1525-2191
AutoresBruno Salaun, Blandine de Saint‐Vis, Nathalie Pacheco, Yves Pachéco, Arnaud Riesler, Sylvie Isaac, Caroline Leroux, Valérie Clair‐Moninot, Jean‐Jacques Pin, Janice Griffith, Isabelle Treilleux, Sophie Goddard, Jean Davoust, Monique J. Kleijmeer, Serge Lebecque,
Tópico(s)Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis
ResumoDendritic cell-lysosomal associated membrane protein (DC-LAMP)/CD208, a member of the lysosomal associated membrane protein (LAMP) family, is specifically expressed by human DCs on activation. However, its mouse counterpart could not be detected in mature DCs. The present study demonstrates that DC-LAMP is constitutively expressed by mouse, sheep, and human type II pneumocytes. Confocal and immunoelectron microscopy showed that mouse DC-LAMP protein co-localizes with lbm180, a specific marker for the limiting membrane of lamellar bodies that contain surfactant protein B, as well as with intracellular MHC class II molecules that accumulate in the same organelles. Expression of DC-LAMP was also occasionally detected at the cell surface of type II pneumocytes. Interestingly, human bronchioloalveolar carcinoma tumor cells, which correspond to transformed type II pneumocytes, express DC-LAMP. Similar observations were made in the Jaagsiekte sheep retrovirus-associated ovine pulmonary adenocarcinoma, a model of human bronchioloalveolar carcinoma. This study establishes that DC-LAMP is constitutively expressed in normal type II pneumocytes. Furthermore, DC-LAMP appears to be a marker of transformed type II pneumocytes as well, an observation that may help the study and the classification of human lung adenocarcinomas. Dendritic cell-lysosomal associated membrane protein (DC-LAMP)/CD208, a member of the lysosomal associated membrane protein (LAMP) family, is specifically expressed by human DCs on activation. However, its mouse counterpart could not be detected in mature DCs. The present study demonstrates that DC-LAMP is constitutively expressed by mouse, sheep, and human type II pneumocytes. Confocal and immunoelectron microscopy showed that mouse DC-LAMP protein co-localizes with lbm180, a specific marker for the limiting membrane of lamellar bodies that contain surfactant protein B, as well as with intracellular MHC class II molecules that accumulate in the same organelles. Expression of DC-LAMP was also occasionally detected at the cell surface of type II pneumocytes. Interestingly, human bronchioloalveolar carcinoma tumor cells, which correspond to transformed type II pneumocytes, express DC-LAMP. Similar observations were made in the Jaagsiekte sheep retrovirus-associated ovine pulmonary adenocarcinoma, a model of human bronchioloalveolar carcinoma. This study establishes that DC-LAMP is constitutively expressed in normal type II pneumocytes. Furthermore, DC-LAMP appears to be a marker of transformed type II pneumocytes as well, an observation that may help the study and the classification of human lung adenocarcinomas. The lysosomal associated membrane protein (LAMP) family consists of a group of heavily glycosylated proteins accounting for approximately half of the protein content in the lysosomal membrane. They all contain a conserved intracytoplasmic tyrosine-based lysosome-targeting motif YXXϕ (where ϕ represents a bulky hydrophobic residue).1Hunziker W Geuze HJ Intracellular trafficking of lysosomal membrane proteins.Bioessays. 1996; 18: 379-389Crossref PubMed Scopus (241) Google Scholar Several members of this family (LAMP-1 to LAMP-3 and CD68) were cloned in a broad range of species.1Hunziker W Geuze HJ Intracellular trafficking of lysosomal membrane proteins.Bioessays. 1996; 18: 379-389Crossref PubMed Scopus (241) Google Scholar Although LAMP-1 and LAMP-2 are ubiquitously expressed,2Furuta K Yang XL Chen JS Hamilton SR August JT Differential expression of the lysosome-associated membrane proteins in normal human tissues.Arch Biochem Biophys. 1999; 365: 75-82Crossref PubMed Scopus (48) Google Scholar CD68 is mainly restricted to monocytes and macrophages.3Holness CL Simmons DL Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins.Blood. 1993; 81: 1607-1613Crossref PubMed Google Scholar The latest human LAMP protein identified, DC-LAMP/CD208, was originally described as a molecule specifically expressed in mature dendritic cells (DCs).4de Saint-Vis B Vincent J Vandenabeele S Vanbervliet B Pin JJ Aït-Yahia S Patel S Mattei MG Banchereau J Zurawski S Davoust J Caux C Lebecque S A novel lysosome associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment.Immunity. 1998; 9: 325-336Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar DC-LAMP appears transiently on DC activation at the limiting membrane of the MHC class II-containing intracellular compartments (MIIC)4de Saint-Vis B Vincent J Vandenabeele S Vanbervliet B Pin JJ Aït-Yahia S Patel S Mattei MG Banchereau J Zurawski S Davoust J Caux C Lebecque S A novel lysosome associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment.Immunity. 1998; 9: 325-336Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 5Barois N de Saint-Vis B Lebecque S Geuze HJ Kleijmeer MJ MHC class II compartments in human dendritic cells undergo profound structural changes upon activation.Traffic. 2002; 3: 894-905Crossref PubMed Scopus (84) Google Scholar involved in MHC class II peptide loading and transport to the cell surface.5Barois N de Saint-Vis B Lebecque S Geuze HJ Kleijmeer MJ MHC class II compartments in human dendritic cells undergo profound structural changes upon activation.Traffic. 2002; 3: 894-905Crossref PubMed Scopus (84) Google Scholar On further maturation, MHC class II molecules and DC-LAMP segregate: MHC class II molecules are translocated to the cell surface membrane, whereas DC-LAMP concentrates in perinuclear lysosomes. On the basis of these observations, it was proposed that DC-LAMP could play a role in the sorting of the MIIC membrane-associated molecules and the transfer of MHC class II molecules to the cell surface.4de Saint-Vis B Vincent J Vandenabeele S Vanbervliet B Pin JJ Aït-Yahia S Patel S Mattei MG Banchereau J Zurawski S Davoust J Caux C Lebecque S A novel lysosome associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment.Immunity. 1998; 9: 325-336Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar Human, monkey, and mouse DC-LAMP mRNAs were shown to be expressed in the lung4de Saint-Vis B Vincent J Vandenabeele S Vanbervliet B Pin JJ Aït-Yahia S Patel S Mattei MG Banchereau J Zurawski S Davoust J Caux C Lebecque S A novel lysosome associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment.Immunity. 1998; 9: 325-336Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 6Ozaki K Nagata M Suzuki M Fujiwara T Ueda K Miyoshi Y Takahashi E Nakamura Y Isolation and characterization of a novel human lung-specific gene homologous to lysosomal membrane glycoproteins 1 and 2: significantly increased expression in cancers of various tissues.Cancer Res. 1998; 58: 3499-3503PubMed Google Scholar, 7Fuller CL Choi YK Fallert BA Capuano III, S Rajakumar P Murphey-Corb M Reinhart TA Restricted SIV replication in rhesus macaque lung tissues during the acute phase of infection.Am J Pathol. 2002; 161: 969-978Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 8Salaun B de Saint-Vis B Clair-Moninot V Pin JJ Barthelemy-Dubois C Kissenpfennig A Peronne C Bates E Mattei MG Lebecque S Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP.Eur J Immunol. 2003; 33: 2619-2629Crossref PubMed Scopus (29) Google Scholar but the cellular source was not determined. Of note, murine DC-LAMP was not detected in mouse DCs.8Salaun B de Saint-Vis B Clair-Moninot V Pin JJ Barthelemy-Dubois C Kissenpfennig A Peronne C Bates E Mattei MG Lebecque S Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP.Eur J Immunol. 2003; 33: 2619-2629Crossref PubMed Scopus (29) Google Scholar In the present study, using specific monoclonal antibodies, we establish that DC-LAMP is specifically expressed by mouse, sheep, and human type II pneumocytes (PnIIs). PnIIs are peripheral pulmonary cells acting as stem cells for repopulation of lung by alveolar type I and type II cells during normal tissue turnover and after injury.9Mason RJ Williams MC Type II alveolar cell. Defender of the alveolus.Am Rev Respir Dis. 1977; 115: 81-91PubMed Google Scholar Beside this progenitor function, PnIIs are also responsible for pulmonary surfactant synthesis, secretion, and recycling.10Fehrenbach H Alveolar epithelial type II cell: defender of the alveolus revisited.Respir Res. 2001; 2: 33-46Crossref PubMed Scopus (581) Google Scholar Detailed analysis of DC-LAMP expression in PnIIs suggests that this molecule may play a role in the conditioning and/or the secretion of surfactant, and that it represents a promising tool for the study of PnIIs and for the diagnosis of lung adenocarcinomas. Commercially available mouse tissue mRNA-loaded membrane (MB no. 2020; OriGene Technologies Inc., Rockville, MD) was hybridized with the full-length cDNA of mouse DC-LAMP labeled by random priming with [32P]-dCTP as described elsewhere.11Valladeau J Clair-Moninot V Dezutter-Dambuyant C Pin JJ Mattéi M-G Ait-Yahia S Bates E Kissenpfennig A Malissen B Koch F Fossiez F Romani N Lebecque S Saeland S Identification of mouse Langerin/CD207 in Langerhans cells and some dendritic cells of lymphoid tissues.J Immunol. 2002; 168: 782-792PubMed Google Scholar Scanning was performed using a PhosporImager (Bio-Rad Laboratories Inc., Hercules, CA). Six-week-old specific pathogen-free BALB/c, 129sv, and C57/BL6 female mice were obtained from Charles River (Iffa Credo, L'Arbresle, France). All mice experiments were done following protocols approved by the institutional animal committee. Lung single-cell suspensions were obtained from manually minced organs after 30 minutes of digestion with 1 mg/ml collagenase (Sigma-Aldrich, St. Louis, MO), crushing through a 0.22-μm cell strainer (BD Labware Falcon, Franklin Lakes, NJ) and final incubation in NH4Cl solution (Stem Cell Technologies, Vancouver, Canada). Lung cells were then either analyzed as total lung cells or subjected to subsequent depletion using rat anti-mouse CD45 monoclonal antibody (mAb) (30-F11; BD Pharmingen, San Diego, CA) and goat anti-rat IgG-coated Dynabeads (Dynal, Oslo, Norway) to enrich the preparation for nonhematopoietic cells. Beads and attached cells were removed with a Dynal magnet. CD45-depleted lung cells were then washed in phosphate-buffered saline (PBS)/0.5 mmol/L ethylenediaminetetraacetic acid (Sigma-Aldrich), spun down, and resuspended either in PBS for cytofluorometric analysis or in serum-free Dulbecco's modified Eagle's medium-F12 (Invitrogen, Carlsbad, CA) for subsequent confocal and immunoelectron microscopy (IEM) studies. Human lung tumor biopsies were obtained from the Centre Hospitalier Lyon Sud tissue collection (Lyon, France). Sheep tissues were obtained postmortem from naturally Jaagsiekte sheep retrovirus (JSRV)-infected animals. Presence of typical lesions associated with ovine pulmonary adenocarcinoma was confirmed by histopathological examination. Rat anti-mouse DC-LAMP mAbs were obtained by immunizing rats with COP5 cells transfected with mouse DC-LAMP, cDNA and hybridoma supernatants were screened by immunocytochemistry and flow cytometry on mouse DC-LAMP-transfected cells, as described in detail elsewhere.8Salaun B de Saint-Vis B Clair-Moninot V Pin JJ Barthelemy-Dubois C Kissenpfennig A Peronne C Bates E Mattei MG Lebecque S Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP.Eur J Immunol. 2003; 33: 2619-2629Crossref PubMed Scopus (29) Google Scholar Balb/C mouse lungs were collected and instilled intratracheally with Cryojung tissue-freezing medium (Leica GmbH, Nussloch, Germany) before snap-freezing in liquid nitrogen. Tissue cryosections (7-μm thick) were fixed in acetone. DC-LAMP single labeling was performed on mouse lung cryosections using standard immunohistochemistry as described elsewhere.8Salaun B de Saint-Vis B Clair-Moninot V Pin JJ Barthelemy-Dubois C Kissenpfennig A Peronne C Bates E Mattei MG Lebecque S Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP.Eur J Immunol. 2003; 33: 2619-2629Crossref PubMed Scopus (29) Google Scholar Double-immunohistofluorescence stainings were performed on cryosections after saturation in PBS/2% bovine serum albumin/10% goat normal serum, using a panel of antibodies [mouse anti-rat lbm180 (3C9; Bender MedSystems Diagnostics GmbH, Vienna, Austria), rat anti-mouse CD44v6 (BMS145, Bender MedSystem), rat anti-mouse I-A/I-E (M5/114, BD Pharmingen) or corresponding isotype-matched control IgG]. Binding was detected with appropriate goat anti-species F(ab)′2 coupled to Alexa 488 (Molecular Probes, Eugene, OR). After incubation in 10% rat serum, rat anti-mouse DC-LAMP antibodies coupled in the laboratory to Alexa 594 or unrelated isotype-matched control IgG were applied. After washes in PBS and water, slides were mounted in Fluoromount (Southern Biotechnology Associates, Birmingham, AL) and analyzed with an Axioskop microscope (Zeiss, Germany). Bouin-fixed, paraffin-embedded sheep and human lung sections were deparaffinized in toluene for 30 minutes, rehydrated in successive baths of alcohol (100% then 95%) and rinsed in water. Antigen retrieval was performed with microwave (15 minutes at 750 W) in citrate buffer (DAKO, Glostrup, Denmark). Endogenous peroxidases were blocked with 0.3% H2O2. After saturation in protein blocking reagent solution (DAKO), staining was performed with rat anti-mouse DC-LAMP-specific mAb (clone 1010E1, which cross-reacts with sheep and human DC-LAMP proteins) diluted in DAKO diluent (DAKO). Revelation was then performed using the Ultratech HRP and Ultratech DAB kits (Immunotech, Marseille, France) according to manufacturer's instructions. Slides were counterstained with Harry's hematoxylin before mounting. CD45-depleted lung cells were resuspended in serum-free medium and allowed to adhere on 12-mm diameter coverslips (Polylabo, Strasbourg, France) precoated with poly-l-lysine (Sigma-Aldrich). After saturation with 10% fetal calf serum-containing medium at 37°C, cells were fixed in 3.7% paraformaldehyde, washed in PBS/10% fetal calf serum/2% bovine serum albumin, then in PBS, and finally permeabilized with PBS/0.05% saponin (Sigma-Aldrich)/0.2% bovine serum albumin. Staining with rat anti-mouse DC-LAMP mAb was performed for 1 hour at 37°C, and detected with goat anti-rat IgG F(ab)′2 coupled to Alexa 594 (Molecular Probes). After saturation with 10% rat serum (DAKO), co-staining was performed with either fluorescein isothiocyanate-coupled rat anti-mouse I-Ad (2G9, BD Pharmingen), or with mouse anti-rat lbm180 (3C9, Bender MedSystem), the latter being detected with goat anti-mouse IgG F(ab)′2 coupled to Alexa 488 (Molecular Probes). Double staining of MHC class II and lbm180 was performed first with rat anti-mouse I-Ad (2G9, BD Pharmingen) detected with goat anti-rat IgG F(ab)′2 coupled to Alexa 594 (Molecular Probes), and after saturation with rat serum, binding of mouse anti-rat lbm180 (3C9, Bender MedSystem) was achieved and detected with goat anti-mouse IgG F(ab)′2 coupled to Alexa 488 (Molecular Probes). After washes in PBS and water, coverslips were mounted onto glass slides in Fluoromount and analyzed with a TSC NT microscope (Leica). CD45-depleted lung cells were fixed in 2% paraformaldehyde/0.2% glutaraldehyde and processed for ultrathin cryosectioning and immunogold labeling as described.12Kleijmeer MJ Posthuma G Geuze HJ Immunoelectron microscopy of antigen processing in dendritic cells.in: Robinson SP Stagg AJ Methods in Molecular Medicine: Dendritic Cell Protocols. Humana Press, Totowa2001: 387-411Crossref Google Scholar Immunogold labeling was performed with the following antibodies: rat anti-mouse DC-LAMP, mouse anti-rat lbm180 (3C9, Bender MedSystems), rat anti-mouse MHC class II I-Ad (2G9, BD Pharmingen), rabbit polyclonal antisera against the cytoplasmic tails of mouse MHC class II α- and β-chain (generous gift from AY Rudensky, Washington University, Seattle, WA) and rabbit anti-bovine mature surfactant protein B.13Voorhout WF Veenendaal T Haagsman HP Weaver TE Whitsett JA van Golde LM Geuze HJ Intracellular processing of pulmonary surfactant protein B in an endosomal/lysosomal compartment.Am J Physiol. 1992; 263: L479-L486PubMed Google Scholar Double-extracellular/intracellular staining on lung cell suspensions was performed using standard techniques. Briefly, cells were incubated with biotin-coupled mAbs and subsequently with R-phycoerythrin (R-PE)-coupled streptavidin (DAKO). After washes in PBS and permeabilization with the Fix and Perm kit (BD Pharmingen), cells were incubated with rat anti-mouse DC-LAMP mAb coupled to Alexa 488 or with isotype-matched negative control. Fluorescence was analyzed with a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). The mouse homologue of human DC-LAMP has recently been cloned from a lung cDNA library because the mRNA had been detected in this tissue by Northern blot using the human DC-LAMP cDNA as a probe.8Salaun B de Saint-Vis B Clair-Moninot V Pin JJ Barthelemy-Dubois C Kissenpfennig A Peronne C Bates E Mattei MG Lebecque S Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP.Eur J Immunol. 2003; 33: 2619-2629Crossref PubMed Scopus (29) Google Scholar In the present study, mouse DC-LAMP mRNA expression was analyzed by Northern blot on commercially available membrane loaded with mRNA from 12 mouse tissues (Figure 1) using the mouse full-length DC-LAMP cDNA as a probe. A strong single 3.2-kb band was detected in the lung, similar to what has been previously observed in human4de Saint-Vis B Vincent J Vandenabeele S Vanbervliet B Pin JJ Aït-Yahia S Patel S Mattei MG Banchereau J Zurawski S Davoust J Caux C Lebecque S A novel lysosome associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment.Immunity. 1998; 9: 325-336Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 6Ozaki K Nagata M Suzuki M Fujiwara T Ueda K Miyoshi Y Takahashi E Nakamura Y Isolation and characterization of a novel human lung-specific gene homologous to lysosomal membrane glycoproteins 1 and 2: significantly increased expression in cancers of various tissues.Cancer Res. 1998; 58: 3499-3503PubMed Google Scholar and mouse.8Salaun B de Saint-Vis B Clair-Moninot V Pin JJ Barthelemy-Dubois C Kissenpfennig A Peronne C Bates E Mattei MG Lebecque S Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP.Eur J Immunol. 2003; 33: 2619-2629Crossref PubMed Scopus (29) Google Scholar No mRNA expression could be detected in any other organ, including brain, heart, kidney, liver, skin, muscles, small intestine, spleen, stomach, testis, and thymus. The absence of DC-LAMP in mouse lymphoid tissues contrasts with human data and was explained by the lack of expression in mature mouse DCs.8Salaun B de Saint-Vis B Clair-Moninot V Pin JJ Barthelemy-Dubois C Kissenpfennig A Peronne C Bates E Mattei MG Lebecque S Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP.Eur J Immunol. 2003; 33: 2619-2629Crossref PubMed Scopus (29) Google Scholar To extend the analysis of mouse DC-LAMP distribution, mouse lung tissue sections were labeled with rat monoclonal antibodies raised against the recombinant mouse protein. A strong signal was detected within a population of peripheral lung cells (Figure 2A1) with no labeling of bronchial epithelium. Both the localization and the cuboidal shape of DC-LAMP-positive cells protruding into the lumina of the alveolae (Figure 2A1, inset) suggested they could correspond to PnIIs. To formally identify the DC-LAMP-expressing cells, double-immunofluorescent labeling was performed on mouse lung cryosections (Figure 2, A2 to A4). Almost all DC-LAMP-positive cells were found to co-express the lamellar body (LB) limiting membrane marker lbm180/ABCA3 (Figure 2A2), thereby confirming that these cells were PnIIs.14Zen K Notarfrancesco K Oorschot V Slot JW Fisher AB Shuman H Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane.Am J Physiol. 1998; 275: L172-L183PubMed Google Scholar, 15Mulugeta S Gray JM Notarfrancesco KL Gonzales LW Koval M Feinstein SI Ballard PL Fisher AB Shuman H Identification of LBM180, a lamellar body limiting membrane protein of alveolar type II cells, as the ABC transporter protein ABCA3.J Biol Chem. 2002; 277: 22147-22155Crossref PubMed Scopus (187) Google Scholar Most DC-LAMP+ cells were also shown to express the v6 isoform of the CD44 antigen (Figure 2A3), which is specific in the lung for bronchial epithelium and alveolar type II cells.16Kasper M Gunthert U Dall P Kayser K Schuh D Haroske G Muller M Distinct expression patterns of CD44 isoforms during human lung development and in pulmonary fibrosis.Am J Respir Cell Mol Biol. 1995; 13: 648-656Crossref PubMed Scopus (55) Google Scholar Thus, the vast majority of PnIIs in normal noninflammatory lung express DC-LAMP, and this constitutive expression is in striking contrast with the tight regulation described in human DCs.4de Saint-Vis B Vincent J Vandenabeele S Vanbervliet B Pin JJ Aït-Yahia S Patel S Mattei MG Banchereau J Zurawski S Davoust J Caux C Lebecque S A novel lysosome associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment.Immunity. 1998; 9: 325-336Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar PnIIs have previously been reported to express MHC class II molecules and to be able to present antigens to CD4+ T cells in vitro.17Steiniger B Sickel E Class II MHC molecules and monocytes/macrophages in the respiratory system of conventional, germ-free and interferon-gamma-treated rats.Immunobiology. 1992; 184: 295-310Crossref PubMed Scopus (14) Google Scholar, 18Cunningham AC Milne DS Wilkes J Dark JH Tetley TD Kirby JA Constitutive expression of MHC and adhesion molecules by alveolar epithelial cells (type II pneumocytes) isolated from human lung and comparison with immunocytochemical findings.J Cell Sci. 1994; 107: 443-449PubMed Google Scholar, 19Christensen PJ Armstrong LR Fak JJ Chen GH McDonald RA Toews GB Paine III, R Regulation of rat pulmonary dendritic cell immunostimulatory activity by alveolar epithelial cell-derived granulocyte macrophage colony-stimulating factor.Am J Respir Cell Mol Biol. 1995; 13: 426-433Crossref PubMed Scopus (40) Google Scholar Double labeling of lung tissue sections revealed a heterogeneous MHC class II molecule expression in ∼50% of DC-LAMP+ cells (Figure 2A4). Of note, MHC class II-positive cells not staining for DC-LAMP likely represented alveolar macrophages and/or peripheral DCs. Further characterization of DC-LAMP+ lung cells was obtained by flow cytometry analysis (Figure 2B). The proportion of DC-LAMP+ cells among the total cells recovered after lung digestion varied from 1 to 10% (mean value, 3.8%). The lack of expression of CD45 (leukocyte common antigen restricted to hematopoietic cells), CD11b, and CD11c (leukocyte-restricted integrins) by DC-LAMP+ cells clearly establishes that they are neither DCs nor alveolar macrophages. Instead, all DC-LAMP+ cells co-expressed high levels of CD54 (ICAM-1) and MHC class II molecules, in agreement with previous reports on PnIIs.17Steiniger B Sickel E Class II MHC molecules and monocytes/macrophages in the respiratory system of conventional, germ-free and interferon-gamma-treated rats.Immunobiology. 1992; 184: 295-310Crossref PubMed Scopus (14) Google Scholar, 18Cunningham AC Milne DS Wilkes J Dark JH Tetley TD Kirby JA Constitutive expression of MHC and adhesion molecules by alveolar epithelial cells (type II pneumocytes) isolated from human lung and comparison with immunocytochemical findings.J Cell Sci. 1994; 107: 443-449PubMed Google Scholar, 20Guzman J Izumi T Nagai S Costabel U ICAM-1 and integrin expression on isolated human alveolar type II pneumocytes.Eur Respir J. 1994; 7: 736-739Crossref PubMed Scopus (36) Google Scholar The lower proportion (∼50%) of DC-LAMP+ PnIIs found to express MHC class II molecules by immunohistological analysis likely reflects the difference of sensitivity between the two methods. Furthermore, flow cytometry analysis also showed that half of the DC-LAMP+ population expressed CD95 (Fas antigen), in agreement with previous reports on PnIIs.21Fine A Anderson NL Rothstein TL Williams MC Gochuico BR Fas expression in pulmonary alveolar type II cells.Am J Physiol. 1997; 273: L64-L71PubMed Google Scholar Similar results were obtained with the three strains of mice tested (data not shown). In conclusion, the immunohistological and flow cytometry analysis performed to characterize the DC-LAMP+ lung cells were in agreement with the established PnII phenotype. In human maturing DCs, DC-LAMP has been shown to accumulate at the limiting membrane of the lysosomal compartment devoted to the intracellular storage of MHC class II molecules. To determine the subcellular localization of the protein in PnIIs, DC-LAMP+ cells enriched from total mouse lung single-cell suspensions were analyzed by CLM (Figure 3). CD45-depleted cell preparations were immobilized onto poly-l-lysine slides and stained with anti-DC-LAMP and anti-lbm180 antibodies. Anti-lbm180 mAb recognizes lbm180/ABCA3, an ABC transporter specifically present on the limiting membrane of PnII LBs.14Zen K Notarfrancesco K Oorschot V Slot JW Fisher AB Shuman H Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane.Am J Physiol. 1998; 275: L172-L183PubMed Google Scholar, 15Mulugeta S Gray JM Notarfrancesco KL Gonzales LW Koval M Feinstein SI Ballard PL Fisher AB Shuman H Identification of LBM180, a lamellar body limiting membrane protein of alveolar type II cells, as the ABC transporter protein ABCA3.J Biol Chem. 2002; 277: 22147-22155Crossref PubMed Scopus (187) Google Scholar A partial co-localization of both molecules was observed in intracellular ring-like structures, identified as LBs according to the presence of specific marker lbm180. Two studies14Zen K Notarfrancesco K Oorschot V Slot JW Fisher AB Shuman H Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane.Am J Physiol. 1998; 275: L172-L183PubMed Google Scholar, 22Yamano G Funahashi H Kawanami O Zhao LX Ban N Uchida Y Morohoshi T Ogawa J Shioda S Inagaki N ABCA3 is a lamellar body membrane protein in human lung alveolar type II cells.FEBS Lett. 2001; 508: 221-225Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar have reported a patchy surface expression of lbm180 that corresponds to recent sites of LB fusion with the plasma membrane, leading to the release of surfactant into the alveolar space. Figure 3, C1 to C4, illustrates such an exocytosis event, where LB co-expressing DC-LAMP and lbm180 has fused with the cell surface membrane. Both molecules are also observed to form adjacent patches at the cell surface (Figure 3E, arrows). The subcellular localization of DC-LAMP was further illustrated by IEM. In agreement with the CLM data, DC-LAMP was primarily detected at the limiting membrane of LB (Figure 4A), where it co-localized with lbm180 (Figure 4B). The nature of these organelles expressing DC-LAMP at their limiting membrane was confirmed by double labeling with surfactant protein B (SP-B) (Figure 4C). Furthermore, a low density of DC-LAMP signal was detected in endosomal multivesicular bodies (Figure 4C) and at the cell surface membrane (Figure 4D). Immunohistological and flow cytometry analysis showed expression of both DC-LAMP and MHC class II molecules by PnIIs. Considering the tight association of both molecules within the MIIC of human maturing DCs,4de Saint-Vis B Vincent J Vandenabeele S Vanbervliet B Pin JJ Aït-Yahia S Patel S Mattei MG Banchereau J Zurawski S Davoust J Caux C Lebecque S A novel lysosome associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment.Immunity. 1998; 9: 325-336Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar their relative localizations in PnIIs were compared (Figure 5, Figure 6). CLM analysis revealed a heterogeneous staining, with both single- and double-positive cells. However, in cells expressing both molecules, MHC class II molecules were present not only at the cell surface (Figure 5; A to D), but occasionally co-localized with DC-LAMP in intracellular compartments (Figure 5, A and B) that also contained lbm180 (Figure 5, C and D) and correspond therefore to LB. Of note, MHC class II molecules appear to co-localize more extensively with ABCA3 than DC-LAMP. This suggests that the vesicular membranes and the Golgi cis-ternae stained with anti-lbm180 mAb14Zen K Notarfrancesco K Oorschot V Slot JW Fisher AB Shuman H Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane.Am J Physiol. 1998; 275: L172-L183PubMed Google Scholar also contain MHC class II molecules, but not detectable amounts of DC-LAMP.Figure 6PnIIs express MHC class II molecules at their surface and at the limiting membrane of lamellar bodies. A: Double-immunogold labeling of an isolated mouse lung cell ultrathin cryosection showed the presence of MHC class II on the basolateral plasma membrane (PM) and in lamellar bodi
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