Human Dermal Dendritic Cells Process and Present Soluble Protein Antigens
1998; Elsevier BV; Volume: 110; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.1998.00189.x
ISSN1523-1747
AutoresFrank O. Nestle, Luis Filgueira, Brian J. Nickoloff, Günter Burg,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoRecently, a novel type of dendritic antigen-presenting cell has been identified in the dermis of normal human and mouse skin. These dermal dendritic cells (DDC) occur in higher numbers than epidermal Langerhans cells, represent a distinct differentiation pathway of dendritic cells, and are as potent as Langerhans cells in the activation of superantigen specific T cells. As yet, nothing is known about their capacity to take up, process, and present soluble protein antigens. We used the model of tetanus toxoid (TT) driven T cell proliferation to address these questions. To test for active internalization of TT protein, gold labeled TT was incubated with Langerhans cells and DDC and could be traced to multivesicular endo-lysosomal compartments. DDC internalize TT through a receptor-mediated, clathrin-independent pathway, whereas Langerhans cells predominantly use macropinocytosis. To verify that DDC process TT by the exogenous pathway of antigen presentation, we pulsed DDC with TT protein or TT peptide after preincubation with chloroquine. Preincubation with chloroquine diminished the capacity of DDC to induce TT protein specific T cell proliferation (70–80%), but was not effective to suppress TT peptide induced T cell responses. DDC were as potent as Langerhans cells and 5–10 × more potent than plastic adherent monocytes in the presentation of TT to autologous resting T cells. Furthermore, as few as 50 DDC (stimulator:responder ratio of 1:1000) were able to induce a significant TT specific T cell proliferation. Because a subpopulation of DDC expresses low levels of CD1a, a phenotypic marker of Langerhans cells, sorting of CD1a positive and negative DDC was performed. On a per cell basis, CD1a positive and negative DDC were equally potent at mediating TT specific T cell proliferation. Thus, DDC are able to internalize, process, and present soluble protein antigens such as TT and may therefore play an important role in the regulation of skin immune responses. Recently, a novel type of dendritic antigen-presenting cell has been identified in the dermis of normal human and mouse skin. These dermal dendritic cells (DDC) occur in higher numbers than epidermal Langerhans cells, represent a distinct differentiation pathway of dendritic cells, and are as potent as Langerhans cells in the activation of superantigen specific T cells. As yet, nothing is known about their capacity to take up, process, and present soluble protein antigens. We used the model of tetanus toxoid (TT) driven T cell proliferation to address these questions. To test for active internalization of TT protein, gold labeled TT was incubated with Langerhans cells and DDC and could be traced to multivesicular endo-lysosomal compartments. DDC internalize TT through a receptor-mediated, clathrin-independent pathway, whereas Langerhans cells predominantly use macropinocytosis. To verify that DDC process TT by the exogenous pathway of antigen presentation, we pulsed DDC with TT protein or TT peptide after preincubation with chloroquine. Preincubation with chloroquine diminished the capacity of DDC to induce TT protein specific T cell proliferation (70–80%), but was not effective to suppress TT peptide induced T cell responses. DDC were as potent as Langerhans cells and 5–10 × more potent than plastic adherent monocytes in the presentation of TT to autologous resting T cells. Furthermore, as few as 50 DDC (stimulator:responder ratio of 1:1000) were able to induce a significant TT specific T cell proliferation. Because a subpopulation of DDC expresses low levels of CD1a, a phenotypic marker of Langerhans cells, sorting of CD1a positive and negative DDC was performed. On a per cell basis, CD1a positive and negative DDC were equally potent at mediating TT specific T cell proliferation. Thus, DDC are able to internalize, process, and present soluble protein antigens such as TT and may therefore play an important role in the regulation of skin immune responses. dermal dendritic cells dendritic cells tetanus toxoid Dendritic cells (DC) belong to a family of antigen-presenting cells (APC) that are located in the T cell dependent area of lymphoid tissues and at the barrier zones where antigen entry into the human body through the skin, lung, and gut may take place. The best studied DC in the human system is the Langerhans cell, located in a suprabasal position in human and murine epidermis (Stingl and Bergstresser, 1995Stingl G. Bergstresser P.R. Dendritic cells: a major story unfolds.Immunol Today. 1995; 16: 330-333Abstract Full Text PDF PubMed Scopus (100) Google Scholar). Recently, the dermis has been appreciated as an important immunoreactive site (Streilein, 1993Streilein J.W. Dermal cells: Underappreciated components of skin-associated lymphoid tisuue (SALT).in: Nickoloff B.J. Dermal Immune System. CRC Press, Boca Raton1993: 25-38Google Scholar). The understanding of components of the dermal immune system may be important not only for most inflammatory and neoplastic skin diseases, but also for the understanding of mechanisms involved in anti-tumor vaccination (Gross, 1943Gross L. Intradermal immunization of C3H mice against a sarcoma that originated in the same line.Cancer Res. 1943; 3: 326-333Google Scholar). A new member of the DC family has been described in a perivascular location at the strategic dermal interface between the blood supply and the epidermis (Headington, 1986Headington J.T. The dermal dendrocyte.in: Callen J.P. Dahl H.V. Golitz L.E. Rasmussen J.E. Stegman S.T. Advances in Dermatology. Yearbook Medical Publishers, Chicago1986: 159-170Google Scholar; Lenz et al., 1993Lenz A. Heine M. Schuler G. Romani N. Human and murine dermis contain dendritic cells.J Clin Invest. 1993; 92: 2587-2596Crossref PubMed Scopus (252) Google Scholar; Meunier et al., 1993Meunier L. Gonzales-Ramos A. Cooper K.D. Heterogenous populations of class II MHC+ cells in human dermal cell suspensions.J Immunol. 1993; 151: 4067-4080PubMed Google Scholar; Nestle et al. 1993; Sepulveda-Merrill et al., 1994Sepulveda-Merrill C. Mayall S. Hamblin A.S. Breathnach S.M. Antigen-presenting capacity in normal human dermis is mainly subserved by CD1a + cells.Br J Dermatol. 1994; 131: 15-22Crossref PubMed Scopus (26) Google Scholar). Recent evidence indicates a role for dermal DC in contact hypersensitivity reactions (Streilein, 1989Streilein J.W. Antigen-presenting cells in the induction of contact hypersensitivity in mice: evidence that Langerhans cells are sufficient but not required.J Invest Dermatol. 1989; 93: 443-448Crossref PubMed Scopus (73) Google Scholar; Tse and Cooper, 1990Tse H. Cooper K.D. Cutaneous dermal Ia+ cells are capable of initiating delayed type hypersensitivity responses.J Invest Dermatol. 1990; 94: 267Abstract Full Text PDF PubMed Google Scholar) and in diseases such as psoriasis (Nestle et al., 1994Nestle F.O. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells in psoriasis. Autostimulation of T lymphocytes and induction of Th1 type cytokines.J Clin Invest. 1994; 94: 202-209Crossref PubMed Google Scholar), cutaneous lymphoproliferative disorders (Nestle and Nickoloff, 1994Nestle F.O. Nickoloff B.J. Role of dendritic cells in benign and malignant lymphocytic infiltrates of the skin.Dermatol Clin. 1994; 12: 271-282PubMed Google Scholar), and dermatofibroma (Nestle et al., 1995Nestle F.O. Nickoloff B.J. Burg G. Dermatofibroma: an abortive immunoreactive process mediated by dermal dendritic cells?.Dermatol. 1995; 190: 265-268Crossref PubMed Scopus (64) Google Scholar), as well as Kaposis' sarcoma (Nickoloff and Griffith, 1989Nickoloff B.J. Griffith C.E.M. Factor XIIIa expressing dermal dendrocytes are increased in AIDS-associated Kaposi's sarcoma.Science. 1989; 243: 1736Crossref PubMed Scopus (83) Google Scholar). More recently, two major pathways of DC differentiation have been described: one pathway leading to the development of Langerhans cell-like cells, and a second pathway where dermal dendritic cells (DDC) seem to be the prototypic cell type (Caux et al., 1996Caux C. Vanbervliet B. Massacrier C. et al.CD34+ hematopoietic progenitors from human cord blood diffeerntiate along two independent dendritic cell pathways in response to GM-CSF and TNFa.J Exp Med. 1996; 184: 695-706Crossref PubMed Scopus (810) Google Scholar). Therefore, from the comparative study of Langerhans cells and DDC one may learn not only about the skin immune system, but also about the development of DC in general. DDC have been extensively studied concerning their phenotype and potency for presentation of bacterial derived superantigens to T cells (Nestle and Nickoloff, 1995Nestle F.O. Nickoloff B.J. Dermal dendritic cells are important members of the skin immune system.Adv Exp Med Biol. 1995; 378: 111-116Crossref PubMed Scopus (30) Google Scholar). In contrast, no data are available regarding phagocytosis, processing, and presentation of more widespread soluble protein antigens. We have investigated these issues using tetanus toxoid (TT) as a model antigen because this system is well defined (Demotz et al., 1989Demotz S. Matricardi P.M. Irle C. Panina P. Lanzavecchia A. Corradin G. Processing of tetanus toxin by human antigen-presenting cells.J Immunol. 1989; 143: 3881-3886PubMed Google Scholar), and circulating TT specific memory T cells are frequent in the European population. In this study we demonstrate that DDC effectively take up TT through a receptor mediated nonclathrin dependent mechanism and transfer it to multilaminar endo-lysosomal compartments. DDC are as potent as Langerhans cells and more potent than monocytes in presenting TT protein to autologous CD4 T cells. Furthermore, on a per cell basis, CD1a positive and negative DDC subsets have similar capacities of antigen presentation. RPMI 1640 (Gibco, Basel, Switzerland) was supplemented with 10% fetal calf serum (Seromed, Basel, Switzerland), 10 U penicillin/streptomycin per ml (Gibco), and 50 μg gentamycin per ml (Gibco). TT was obtained from the Swiss Serum Institute (Berne, Switzerland). Synthetic TT derived peptide p30 (TT 947–967; FNNFTVSFWLRVPKVSASHLE) was obtained from G. Corradin (University of Lausanne, Switzerland). TT-gold complexes were prepared as already described (Filgueira et al., 1989Filgueira L. Groscurth P. Aguet M. Binding and internalization of gold-labeled IFN-g by human RAJI cells.J Immunol. 1989; 142: 3336-3339Google Scholar). Briefly, colloidal gold particles, 4 nm in size, and TT protein were adjusted at pH 6.1 (isoelectric point) and mixed by stirring. TT-gold complex was purified by ultracentrifugation (45 min × 100,000 ×g). Specificity and antigenic properties of TT-gold were tested using TT specific FC4-EBV cell clone (kindly provided by A. Lanzavecchia, Basel Institute of Immunology, Switzerland) and TT specific cell lines (data not shown). TT-gold was found to be equivalent to soluble TT-protein concerning binding to FC4 cells and activation of TT specific T cells. Fluoroscein isothiocyanate conjugated isotype controls were purchased from Becton-Dickinson (Mountain View, CA). CD1a fluoroscein isothiocyanate (OKT6) antibody was purchased from Ortho Diagnostics (Raritan, NJ). Normal human skin was obtained from corrective plastic surgery of breast and abdomen. The inclusion criterion was a recent history of tetanus vaccination. Split-thickness skin was prepared using a dermatome (Aesculap, Tuttlingen, Germany) and than incubated in a solution of the bacterial protease dispase type 2 (Boehringer, Mannheim, Germany) at a final concentration of 1.2 U RPMI 1640 per ml at 4°C overnight. After the incubation period, the epidermis and dermis could be easily separated. Epidermal and dermal sheets were then rinsed several times in phosphate-buffered saline, cut into small pieces (≈1–10 mm), and placed in RPMI 1640 supplemented with 10% fetal calf serum, in 10 cm tissue culture plates (Merck, Dietikon, Switzerland). After 2–3 d the pieces of tissue were removed and the medium was collected. The cells that had migrated out of the tissue sections into the medium were spun, resuspended in 1–2 ml fresh medium, and stained with trypan blue. Cell viability and cell number was assessed with a hemocytometer. Further enrichment of DDC and Langerhans cells was achieved through separation with a metrizamide gradient as described (Nestle et al. 1993). Briefly, cells were layered onto 3 ml columns of hypertonic 14.5% metrizamide (Nycomed Pharma AS, Oslo, Norway) and sedimented at 650 ×g for 10 min at room temperature. Low density interphase cells were collected and washed in two successive less hypertonic washes to return cells to isotonicity. DDC expressed high levels of HLA DR, CD80, CD86, ICAM-1, and CD83 (HB15) in a homogeneous fashion (data not shown). For some experiments, metrizamide low density fractions were further enriched using sterile cell sorting with a FACS Star Plus flow cytometer (Becton-Dickinson, San Jose, CA). The cells were incubated with a fluoroscein isothiocyanate conjugated CD1a monoclonal antibody for 30 min on ice and then washed three times. CD1a positive and negative DDC were obtained after sorting with a purity of > 95%. Highly purified resting CD4 positive T cells were obtained by high gradient magnetic cell sorting using the MACS technique according to the manufacturer's procedure (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, human PBMC were enriched by Ficoll-Hypaque gradients. T lymphocytes were subsequently obtained by a first round of negative selection of HLA-DR positive cells, followed by positive selection for CD4 positive T cells. T cells purified by this procedure were always more than 98% CD2+/CD4+. No HLA-DR positive T cells were detected by fluorescence-activated cell sorter (FACS) analysis. Cells were prefixed with 2.5% glutaraldehyde + 0.8% paraformaldehyde, and postfixed with an aqueous solution of 1% OsO4 containing 1.5% K4(FeCN)6. Subsequently, the specimens were dehydrated in an alcohol serie and embedded into epon. Ultrathin sections (≈50 nm) were contrasted with lead citrate and uranyl acetate and examined with a TEM 420 (Phillips, Eindhoven, the Netherlands). All antibody dilutions and washes were in phosphate-buffered saline containing 1% bovine serum albumin (Sigma, Buchs, Switzerland). Negative controls to rule out nonspecific staining or Fc receptor mediated binding of antibodies were unrelated monoclonal antibodies of the same isotype. Cells were aliquoted at 1 × 105 per reaction and all staining was performed for 25 min at 4°C using the indicated primary reagents. Analysis was performed by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA) equipped with a Lysis 2 software. Proliferation assays were performed using standard techniques. Briefly, for the TT specific T cell proliferation assays, gamma irradiated stimulator cells (3000 rad) were cocultured at the indicated concentrations with 5 × 104 purified CD4 positive T cells in 96 well round-bottom culture plates (Falcon-Becton-Dickinson, Basel, Switzerland) in 200 μl RPMI 1640 + 10% fetal calf serum for 3 d. Cells were pulsed for the final 18 h of the incubation period with 1 μCi per well of [3H]TdR (Amersham, Little Chielfont, U.K.). Values are expressed as the mean cpm ± SD of triplicate wells. Few data are available regarding the internalization of soluble protein antigens in human Langerhans cells (Schuler et al., 1991Schuler G. Romani N. Stössel H. Wolff K. Structural organization and biological properties of Langerhans cells.in: Schuler G. Epidermal Langerhans Cells. CRC Press, Boca Raton1991: 87-137Google Scholar) and no data are available for antigen uptake of DDC. To ensure that TT is taken up by the APC, and to visualize the endocytic and processing pathway, Langerhans cells and DDC were studied with TEM. For that purpose, the cells were incubated at culture conditions with gold (4 nm) labeled TT (TT-gold) for different time periods before they were processed for standard TEM. To demonstrate that TT-gold was functional, Langerhans cells or DDC were pulsed for 2 h at 37°C with TT-gold or TT at a concentration of 10 μg per ml and incubated with autologous CD4 T cells. Responding T cells proliferated equally well to TT-gold or TT (data not shown). Langerhans cells and DDC demonstrated a similar ultramorphology with typical membrane processes or lamellipodia (Figures 1a, 2a). The cytoplasm contained few lysosomes, many mitochondria, profiles of smooth endoplasmatic reticulum, and many electron-lucent vacuoles. Differences concerning uptake of TT-gold were detected between Langerhans cells and DDC. DDC bound TT-gold at their surface membrane before endocytosis took place Figure 2b. Also, in the endosomes, TT-gold was found to be bound to the vesicular membrane Figure 2c. In contrast, no membrane binding of TT-gold was found in the case of Langerhans cells, either to the surface Figure 1b or to the endosomal membrane Figure 1c. In the endosome of Langerhans cells, TT-gold was laying without contact to the vesicle membrane free in the vesicular lumen Figure 1c. In both cell types endocytic vesicles were uncoated and were of various different sizes (100–300 nm diameter). Towards the lysosomal compartment, the gold particle bearing endosomes developed a lamellar or multivesicular substructure in Langerhans cells Figure 1d and DDC Figure 2d, which corresponds to the ultramorphology of major histocompatibility complex (MHC) class II loading compartments.Figure 2Ultrastructural analysis of TT uptake and processing in DDC. (A) Overview of a DDC with typical dendritic membrane protrusions. (B) Colloidal gold (4 nm) labeled TT (arrowhead) bound to the surface membrane of a DDC. (C) TT-gold complexes (arrowhead) are visualized bound to the membrane of endocytotic vesicles near to the cell surface of a DDC. (D) TT-gold complexes (arrowhead) are visualized bound to membrane structures in lamellar vesicles in the area of the early lysosomal compartment.View Large Image Figure ViewerDownload (PPT) We used TT as a model antigen because many human beings have a history of vaccination for TT and possess circulating TT specific memory T cells. To test if DDC present TT to T cells, they were compared with Langerhans cells and plastic adherent monocytes from the same donor, using 5 × 104 autologous resting CD4 positive T cells as responders. The T cell responder population was greater than 98% pure and devoid of accessory cells as confirmed by nonresponsiveness to phytohemagglutinin. Langerhans cells, DDC, and monocytes were pulsed with TT for 2 h at 37°C, washed extensively, and incubated with 5 × 104 responder cells (stimulator:responder ratio of 1:1000, 1:100, and 1:10, respectively). DDC were as effective as Langerhans cells in presenting TT to autologous T cells and 5–10 times more potent than monocytes Figure 3. Furthermore, as few as 50 DDC were able to induce a significant TT specific T cell proliferation (> five times background proliferation). To verify if DDC process TT by the exogenous (acidic endosomal) pathway of antigen presentation, we studied the antigen-processing ability in the presence of chloroquine, a selective inhibitor of this pathway (Streicher et al., 1984Streicher H.Z. Berkower I.J. Busch M. Gurd F.R.N. Berzofsky J.A. Antigen confirmation determines processing requirements for T-cell activation.Proc Natl Acad Sci. 1984; 81: 6831-6835Crossref PubMed Scopus (145) Google Scholar). Langerhans cells and DDC were pulsed with TT protein (TTprot at 10 μg per ml) or a universally immunogenic TT peptide p30 (Panina-Bordignon et al., 1989Panina-Bordignon P. Tan A. Termitjelen A. Demotz S. Corradin G. Lanzavecchia A. Universally immunogenic T cell epitopes: promiscuous binding to human MHC class II and promiscuous recognition by T cells.Eur J Immunol. 1989; 19: 2237-2242Crossref PubMed Scopus (620) Google Scholar) (TTpept at 10 ng per ml) in the presence or absence of 300 μM chloroquine. Five × 102 APC were then incubated with autologous resting CD4 positive T cells at a stimulator:responder ratio of 1:100 and the proliferative response was tested. TTpept induced T cell responses were used to control for chloroquine induced nonspecific suppression of T cell proliferation. Chloroquine treatment reduced the stimulatory potency of TTprot pulsed DDC and Langerhans cells significantly (68% and 64%, respectively) Figure 4. Using TTpept pulsed APC, no relevant inhibition of T cell stimulation occurred (DDC, 9%; Langerhans cells, 27%) Figure 4. When chloroquine was added to the APC after antigen pulsing, there was no significant inhibition of T cell proliferation (data not shown). Subpopulations of CD1a+ and CD1a– DDC have been described (Meunier et al., 1993Meunier L. Gonzales-Ramos A. Cooper K.D. Heterogenous populations of class II MHC+ cells in human dermal cell suspensions.J Immunol. 1993; 151: 4067-4080PubMed Google Scholar; Nestle et al., 1994Nestle F.O. Zheng X.G. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets [published erratum appears in J Immunol 152(1):376, 1994]..J Immunol. 1993; 151: 6535-6545PubMed Google Scholar). To test the possibility that these subsets differ in their capacity to present antigen, CD1a+ and CD1a– DDC were differentially sorted on a flow cytometer (purity > 95%). They were pulsed with TT (10 μg per ml, 2 h at 37°C) and incubated at different stimulator:responder ratios with autologous resting CD4+ T cells. TT induced T cell proliferation did not significantly differ between the CD1a+ and CD1a– DDC and was comparable with unsorted DDC induced T cell proliferation Figure 5. This indicates that on a per cell basis, CD1a positive and negative DDC are equally able to process and present TT protein to autologous helper T cells. Regional immunity is becoming an increasingly important topic regarding the peripheral activation and de-activation of circulating T cells at various tissue sites (Arnold et al., 1992Arnold B. Schönrich G. Hämmerling G.J. Extrathymic T-cell selection.Curr Opin Immunol. 1992; 4: 166-170Crossref Scopus (14) Google Scholar). In this context, activation of T cells by APC of the dermis that are located at the interface between blood supply and the skin immune system may play a central role in vaccination and disease (Streilein, 1993Streilein J.W. Dermal cells: Underappreciated components of skin-associated lymphoid tisuue (SALT).in: Nickoloff B.J. Dermal Immune System. CRC Press, Boca Raton1993: 25-38Google Scholar). A new member of the DC family of APC has been recently isolated from human and murine dermis and characterized regarding phenotype and T cell stimulatory capacity (Nestle and Nickoloff, 1995Nestle F.O. Nickoloff B.J. Dermal dendritic cells are important members of the skin immune system.Adv Exp Med Biol. 1995; 378: 111-116Crossref PubMed Scopus (30) Google Scholar). Interest in the biology of DDC also stems from the fact that they may be the physiologic representative of a major DC differentiation pathway (Caux et al., 1996Caux C. Vanbervliet B. Massacrier C. et al.CD34+ hematopoietic progenitors from human cord blood diffeerntiate along two independent dendritic cell pathways in response to GM-CSF and TNFa.J Exp Med. 1996; 184: 695-706Crossref PubMed Scopus (810) Google Scholar). No information is currently available concerning uptake, processing, and presentation to T cells of common soluble protein antigens by these cells. We have investigated these issues using TT as a model antigen because this system is well defined (Demotz et al., 1989Demotz S. Matricardi P.M. Irle C. Panina P. Lanzavecchia A. Corradin G. Processing of tetanus toxin by human antigen-presenting cells.J Immunol. 1989; 143: 3881-3886PubMed Google Scholar) and circulating TT specific memory T cells are frequent in the European population. Before immunogenic peptides are presented on surface MHC class II molecules to T cells, protein antigens have to be phagocytosed and processed intracellularily in the endosomal compartment (Germain, 1994Germain R.N. MHC-dependent antigen processing and peptide presentation: providing ligands for T lymphocyte activation.Cell. 1994; 76: 287-299Abstract Full Text PDF PubMed Scopus (1240) Google Scholar). The processing pathway can be visualized by use of gold labeled proteins with TEM (Peters et al., 1995Peters P.J. Rapso G. Neefjes J.L. Oorschot V. Leijendekker R.L. Geuze H.J. Ploegh H.L. Major histocompatibility complex class II compartments in human B lymphoblastoid cells are distinct from early endosomes.J Exp Med. 1995; 182: 325-334Crossref PubMed Scopus (116) Google Scholar). In this study we used gold labeled TT that had been shown to be functionally active concerning activation of TT specific T cells by TT pulsed APC. There was efficient binding of TT-gold to the surface membrane of DDC. The chemical structure of the molecules binding TT-gold has not been determined yet. Despite a receptor mediated uptake of TT-gold that seems to take place in the case of DDC, the endosomal vesicles were uncoated, indicating a clathrin-independent pinocytic pathway (Guagliardi et al., 1990Guagliardi L.E. Koppelman B. Blum J.S. Marks M.S. Cresswell P. Brodsky F.M. Co-localization of molecules involved in antigen processing and presentation in an early endocytic compartment.Nature. 1990; 343: 133-139Crossref PubMed Scopus (297) Google Scholar; Lamaze and Schmid, 1995Lamaze C. Schmid S.L. The emergence of clathrin-independent pinocytic pathways.Curr Opin Cell Biol. 1995; 7: 573-580Crossref PubMed Scopus (249) Google Scholar). This pathway may substitute for clathrin-dependent endocytosis in DDC and is currently under investigation. In contrast, Langerhans cells did not bind TT-gold to the surface membrane, but were still able to take up TT-gold efficiently. In Langerhans cells the endosomal vesicles were also uncoated and displayed typical characteristics of macropinocytosis (Hewlett et al., 1994Hewlett L.J. Prescott A.R. Watts C. The coated pit and macropinocytic pathways serve distinct endosome populations.J Cell Biol. 1994; 124: 689-703Crossref PubMed Scopus (307) Google Scholar). In comparison, Langerhans cells and DDC seem to use different phagocytosis mechanisms for TT uptake as visualized by TEM, namely macropinocytosis and receptor mediated phagocytosis, respectively. The different uptake mechanisms support the concept about two independent DC differentiation pathways (Caux et al., 1996Caux C. Vanbervliet B. Massacrier C. et al.CD34+ hematopoietic progenitors from human cord blood diffeerntiate along two independent dendritic cell pathways in response to GM-CSF and TNFa.J Exp Med. 1996; 184: 695-706Crossref PubMed Scopus (810) Google Scholar), which is probably also reflected at the level of antigen uptake. Both uptake mechanisms are known to drive towards a similar Ag processing compartment (Sallusto et al., 1995Sallusto F. Cella M. Danieli C. Lanzavechia A. Dendritic cells use macropinocytosis and the mannnose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products.J Exp Med. 1995; 182: 389-400Crossref PubMed Scopus (2122) Google Scholar). Accordingly, the endo-lysosomal vesicles, where processing and antigen loading takes place, were identical in both Langerhans cells and DDC. These vesicles displayed lamellar or multivesicular substructures, characteristic for so-called MIIC, and have been proposed as the major antigen processing and loading site in DC (Nijman et al., 1995Nijman H.W. Kleijmer M.J. Ossevoort M.A. et al.Antigen capture and major histocompatiblity class II compartments of freshly isolated and cultured human blood dendritic cells.J Immunol. 1995; 182: 163-174Google Scholar). We did not directly address the question of whether those compartments are identical to multivesicular or multilaminar MHC class II enriched compartments, although the ultramorphology of the compartments concentrating TT suggests their presence also in DDC. To ensure that the immunogenic TT peptides, loaded onto MHC molecules, are derived from intracellular processing of TT and not from processing by exogenous proteases in the culture medium, we took advantage of the fact that antigen processing requires an acidic lysosomal compartment. TT pulsing of Langerhans cells and DDC in the presence of chloroquine, which elevates the pH in lysosomal compartments and blocks processing of TT protein, resulted in significant reduction of TT specific T cell proliferation. In contrast, TT peptide induced T cell proliferation, which is not dependent on intracellular processing, was not significantly inhibited by chloroquine. A relationship between the maturational state of DC and their ability to process complex polypeptides such as TT has been mainly shown for mouse Langerhans cells (Schuler and Steinman, 1985Schuler G. Steinman R.M. Murine epidermal langerhans cells mature into potent immunostimulatory dendritic cells in vitro.J Exp Med. 1985; 161: 526-546Crossref PubMed Scopus (848) Google Scholar; Romani et al., 1989aRomani N. Koide S. Crowley M. et al.Presentation of exogenous protein antigens by dendritic cells to T cell cones: intact protein is presented best by immature epidermal Langerhans cells.J Exp Med. 1989; 169: 1169Crossref PubMed Scopus (460) Google Scholar) and was later also suggested for human Langerhans cells (Romani et al., 1989bRomani N. Lenz A. Glassel H. et al.Cultured human Langerhans cells resemble lymphoid dendritic cells in phenotype and function.J Invest Dermatol. 1989; 93: 600-609Abstract Full Text PDF PubMed Google Scholar; Teunissen et al., 1990Teunissen M.B.M. Wormmeester J. Krieg S.R. Peeters P.J. Vogels I.M.C. Kapsenberg M.L. Bos J.D. Human and epidermal Langerhans cells undergo profound morphologic and phenotypic changes during in vitro culture.J Invest Dermatol. 1990; 94: 166-173Abstract Full Text PDF PubMed Google Scholar); however, efficient presentation of TT by human in vitro generated DC (Sallusto and Lanzavecchia, 1994Sallusto F. Lanzavecchia A. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin-4 and downregulated by tumor necrosis factor-alpha.J Exp Med. 1994; 179: 1109-1118Crossref PubMed Scopus (4395) Google Scholar), as well as human mature cultured Langerhans cells (Cohen and Katz, 1992Cohen P.J. Katz S.I. Cultured human Langerhans cells process and present intact protein antigens.J Invest Dermatol. 1992; 99: 331-336Crossref PubMed Scopus (24) Google Scholar), has been demonstrated. In this study, dependence of the maturational state of DDC on efficiency of antigen processing and presentation could not be investigated due to a lack of an appropriate isolation method for "fresh," immature DDC. The maturational status of the DDC preparation used here has been extensively investigated (Nestle et al., 1994Nestle F.O. Zheng X.G. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets [published erratum appears in J Immunol 152(1):376, 1994]..J Immunol. 1993; 151: 6535-6545PubMed Google Scholar). The high surface expression of CD80, CD86, ICAM-1, CD83 (HB15), and MHC class II molecules, as well as the potent activation of allogeneic T cells, indicates a mature state of the DDC preparation used in this study. DDC were as efficient as Langerhans cells at presenting TT to autologous, resting CD4 positive T cells and were 5–10 times more potent than highly enriched plastic adherent monocytes. Furthermore, as few as 50 DDC induced a significant TT specific T cell proliferation. DDC express high levels of MHC class II molecules and costimulatory molecules such as CD80 and C86 in a homogenous manner (Nestle et al., 1994Nestle F.O. Zheng X.G. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets [published erratum appears in J Immunol 152(1):376, 1994]..J Immunol. 1993; 151: 6535-6545PubMed Google Scholar). Strong surface expression of CD1a is considered a phenotypic hallmark of human Langerhans cells. Because a minor subpopulation (10–15%) of DDC expresses low levels of CD1a (Nestle et al., 1994Nestle F.O. Zheng X.G. Thompson C.B. Turka L.A. Nickoloff B.J. Characterization of dermal dendritic cells obtained from normal human skin reveals phenotypic and functionally distinctive subsets [published erratum appears in J Immunol 152(1):376, 1994]..J Immunol. 1993; 151: 6535-6545PubMed Google Scholar), we addressed the question of whether the minor CD1a+ subpopulation accounts for the potent APC capacity of DDC. Therefore, DDC were differentially sorted in CD1a+ and CD1a– cells and tested for the induction of TT specific T cell proliferation. On a per cell basis, CD1a+ as well as CD1a– subpopulations were equally potent as unsorted DDC in the induction of TT driven T cell proliferation. This is in contrast to a published work investigating fresh dermal cell suspensions, where, by indirect reasoning, the major antigen-presenting cell function for allogeneic T cell proliferations was found mainly in the CD1a population (Sepulveda-Merrill et al., 1994Sepulveda-Merrill C. Mayall S. Hamblin A.S. Breathnach S.M. Antigen-presenting capacity in normal human dermis is mainly subserved by CD1a + cells.Br J Dermatol. 1994; 131: 15-22Crossref PubMed Scopus (26) Google Scholar). It is therefore important to mention that, for reasons of cell purity and to assess TT specific T cell proliferation on a per cell basis, our data were obtained with highly enriched cultured DDC that could account for the different findings. In summary, we have provided evidence that DDC are as efficient as Langerhans cells in the internalization, processing, and presentation of soluble protein antigens such as TT to resting T helper cells. Therefore, DDC in their strategic perivascular location in human dermis may play an important role in the activation of skin seeking T cells in response to soluble protein antigens. Supported by grants from the Cancer League Zürich and the Swiss Cancer League to FON. We thank the Department of Plastic Surgery (Director Prof Dr. V.E. Meyer), University of Zurich Medical School for providing tissue samples, Mrs. E. Niederer, Flow Cytometry Core Unit, University of Zurich Medical School, for cell sorting, and Mrs. M. Balzer, Mrs. M. Erni, and Mr. H. Sonderegger for their excellent technical support.
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