ABSTRACT
The phenotypes and functions of endothelial cells (EC), a heterogeneous cell population, vary along the vascular tree and even in the same organ between different vessels. The placenta is an organ with abundant vessels. To enhance further knowledge concerning placenta derived EC, we develop a new method for isolation, purification and culture of these EC. Moreover, in order to investigate the peculiarity of placenta derived EC we compare their phenotypic and functional characteristics with human dermal lymphatic endothelial cells (HDLEC) and human umbilical vein endothelial cells (HUVEC). Freshly isolated placenta derived EC displayed an elongated shape with pale cytoplasm and showed the typical cobblestone pattern of EC but also a swirling pattern when confluent. FISH-analyses of the isolated EC from placentae of male fetus revealed an XY genotype strongly indicating their fetal origin. Characterisation of placenta derived fetal EC (fEC) underlined their blood vessel phenotype by the expression of vWF, Ulex europaeus lectin-1, HLA-class I molecules, CD31, CD34, CD36, CD51/61, CD54, CD62E, CD105, CD106, CD133, CD141, CD143, CD144, CD146, VEGFR-1, VEGFR-2, EN-4, PAL-E, BMA120, Tie-1, Tie-2 and α-Tubulin. In contrast to previous reports the expression of lymphatic markers, like VEGFR-3, LYVE-1, Prox-1 and Podoplanin was consistently negative. Haematopoietic surface markers like CD45 and CD14 were also always negative. Various functional tests (Dil-Ac-LDL uptake, Matrigel assay and TNF-α induced upregulation of CD62E and CD54) substantiated the endothelial nature of propagated fEC. At the ultrastructural level, fEC harboured numerous microvilli, micropinocytic vesicles at their basis, were rich in intermediate filaments and possessed typical Weibel - Palade bodies. In conclusion, the placenta is a plentiful source of fetal, microvascular, blood EC with an expression profile (CD34+, CD133+, VEGFR-2+, CD45-) suggestive of an endothelial progenitor phenotype.
Subject(s)
Antigens, CD/metabolism , Biomarkers/metabolism , Endothelial Cells/cytology , Placenta/blood supply , Vascular Endothelial Growth Factor Receptor-2/metabolism , AC133 Antigen , Adult , Antigens, CD34/metabolism , Cell Culture Techniques , Cells, Cultured , Cytoplasmic Structures/ultrastructure , Dermis/blood supply , Endothelial Cells/metabolism , Endothelial Cells/ultrastructure , Female , Glycoproteins/metabolism , Human Umbilical Vein Endothelial Cells/cytology , Human Umbilical Vein Endothelial Cells/metabolism , Humans , Leukocyte Common Antigens/metabolism , Microvilli/ultrastructure , Peptides/metabolism , Pregnancy , Term BirthABSTRACT
The challenge in observing de novo virus production in human immunodeficiency virus (HIV)-infected dendritic cells (DCs) is the lack of resolution between cytosolic immature and endocytic mature HIV gag protein. To track HIV production, we developed an infectious HIV construct bearing a diothiol-resistant tetracysteine motif (dTCM) at the C terminus of HIV p17 matrix within the HIV gag protein. Using this construct in combination with biarsenical dyes, we observed restricted staining of the dTCM to de novo-synthesized uncleaved gag in the DC cytosol. Co-staining with HIV gag antibodies, reactive to either p17 matrix or p24 capsid, preferentially stained mature virions and thus allowed us to track the virus at distinct stages of its life cycle within DCs and upon transfer to neighboring DCs or T cells. Thus, in staining HIV gag with biarsenical dye system in situ, we characterized a replication-competent virus capable of being tracked preferentially within infected leukocytes and observed in detail the dynamic nature of the HIV production and transfer in primary DCs.
Subject(s)
Dendritic Cells/ultrastructure , Dendritic Cells/virology , HIV/physiology , HIV/ultrastructure , Microscopy, Fluorescence/methodsABSTRACT
The existence of endothelial progenitor cells (EPC) with high cell-cycle rate in human umbilical cord blood has been recently shown and represents a challenging strategy for therapeutic neovascularization. To enhance knowledge for future cellular therapy, we compared the phenotypic, functional and gene expression differences between EPC-derived cells generated from cord blood CD34(+) cells, and lymphatic and macrovascular endothelial cells (EC) isolated from human foreskins and umbilical veins, respectively. Under appropriate culture conditions, EPC developed into fully matured EC with expression of similar endothelial markers as lymphatic and macrovascular EC, including CD31, CD36, von Willebrand factor FVIII, CD54 (ICAM-1), CD105 (endoglin), CD144 (VE-cadherin), Tie-1, Tie-2, VEGFR-1/Flt-1 and VEGFR-2/Flk-1. Few EPC-derived cells became positive for LYVE-1, indicating their origin from haematopoietic stem cells. However they lacked expression of other lymphatic cell-specific markers such as podoplanin and Prox-1. Functional tests demonstrated that the cobblestone EPC-derived cells up-regulated CD54 and CD62E expression in response to TNF-alpha, incorporated DiI-acetylated low-density liproprotein and formed cord- and tubular-like structures with capillary lumen in three-dimensional collagen culture--all characteristic features of the vascular endothelium. Structures compatible with Weibel-Palade bodies were also found by electron microscopy. Gene microarray profiling revealed that only a small percentage of genes investigated showed differential expression in EPC-derived cells and lymphatic EC. Among them were adhesion molecules, extracellular matrix proteins and cytokines. Our data point to the close lineage relationship of both types of vascular cells and support the theory of a venous origin of the lymphatic system.
Subject(s)
Antigens, CD34/metabolism , Antigens, CD/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Gene Expression Profiling , Glycoproteins/metabolism , Lymphatic Vessels/cytology , Peptides/metabolism , Stem Cells/cytology , AC133 Antigen , Cell Differentiation , Cell Shape , Cells, Cultured , Child , Child, Preschool , Down-Regulation , Endothelial Cells/ultrastructure , Fetal Blood/cytology , Humans , Infant , Infant, Newborn , Male , Oligonucleotide Array Sequence Analysis , Phenotype , Stem Cells/metabolism , Stem Cells/ultrastructure , Up-Regulation , Weibel-Palade Bodies/ultrastructureABSTRACT
Langerin is a C-type lectin expressed by a subset of dendritic leukocytes, the Langerhans cells (LC). Langerin is a cell surface receptor that induces the formation of an LC-specific organelle, the Birbeck granule (BG). We generated a langerin(-/-) mouse on a C57BL/6 background which did not display any macroscopic aberrant development. In the absence of langerin, LC were detected in normal numbers in the epidermis but the cells lacked BG. LC of langerin(-/-) mice did not present other phenotypic alterations compared to wild-type littermates. Functionally, the langerin(-/-) LC were able to capture antigen, to migrate towards skin draining lymph nodes, and to undergo phenotypic maturation. In addition, langerin(-/-) mice were not impaired in their capacity to process native OVA protein for I-A(b)-restricted presentation to CD4(+) T lymphocytes or for H-2K(b)-restricted cross-presentation to CD8(+) T lymphocytes. langerin(-/-) mice inoculated with mannosylated or skin-tropic microorganisms did not display an altered pathogen susceptibility. Finally, chemical mutagenesis resulted in a similar rate of skin tumor development in langerin(-/-) and wild-type mice. Overall, our data indicate that langerin and BG are dispensable for a number of LC functions. The langerin(-/-) C57BL/6 mouse should be a valuable model for further functional exploration of langerin and the role of BG.
Subject(s)
Antigens, Surface/genetics , Antigens, Surface/physiology , Islets of Langerhans/cytology , Langerhans Cells/cytology , Lectins, C-Type/genetics , Lectins, C-Type/physiology , Mannose-Binding Lectins/genetics , Mannose-Binding Lectins/physiology , 9,10-Dimethyl-1,2-benzanthracene , Animals , Antigens/metabolism , Blastocyst/metabolism , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Carcinogens , Cell Movement , Cell Physiological Phenomena , Cytoplasmic Granules/metabolism , Dendritic Cells , Dose-Response Relationship, Drug , Electroporation , Embryo, Mammalian/cytology , Flow Cytometry , Genetic Vectors , Immunohistochemistry , Islets of Langerhans/physiology , Kinetics , Lectins/metabolism , Lymph Nodes/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Electron , Models, Genetic , Mutagenesis , Mutation , Neoplasms/chemically induced , Ovalbumin/metabolism , Phenotype , Stem Cells/cytologyABSTRACT
Activins are members of the transforming growth factor-beta (TGF-beta) family and are important for skin morphogenesis and wound healing. TGF-beta1 is necessary for the population of the epidermis with Langerhans cells (LC). However, a role for activin in LC biology is not known. To address this question, we analyzed skin from transgenic mice overexpressing the activin antagonist follistatin in the epidermis. Using immunofluorescence, we observed a striking decrease in the number of LC in the epidermis of transgenic mice in comparison to wild-type mice. Nevertheless, these LC expressed normal levels of major histocompatibility complex (MHC)-class II and Langerin/ CD207 in situ. In explant cultures of whole ear skin the number of dendritic cells (DC), which migrated into the culture medium, was reduced. This reduction was even more pronounced in cultures of epidermal sheets. Virtually all emigrated cutaneous DC displayed typical morphology with cytoplasmic "veils", showed translocation of MHC-class II to the surface membrane, and expressed the maturation marker 2A1. Thus, cutaneous DC from transgenic mice seemed to mature normally. These results demonstrate that overexpression of follistatin in the epidermis affects LC trafficking but not maturation and suggest a novel role of the follistatin-binding partner activin in LC biology.
Subject(s)
Epidermis , Follistatin/metabolism , Langerhans Cells/metabolism , Activins/metabolism , Animals , Antigens, Surface/metabolism , Biomarkers/metabolism , Cell Movement , Cells, Cultured , Epidermal Cells , Epidermis/metabolism , Follistatin/genetics , Genes, MHC Class II , Langerhans Cells/cytology , Lectins, C-Type/metabolism , Mannose-Binding Lectins/metabolism , Mice , Mice, Transgenic , Tissue Culture TechniquesABSTRACT
Langerhans cells of the epidermis and dermal dendritic cells screen the skin for invading antigens. They initiate primary immune responses after migrating from sites of antigen uptake to lymphoid organs. The skin is a feasible model to study the morphology and regulation of dendritic cell migration. We therefore used murine skin explant cultures for tracking the pathways of dendritic cell migration by electron microscopy. Several novel observations are reported. (i) In 48 h cultures of epidermal sheets numerous Langerhans cells migrated out between keratinocytes extending long and thin cytoplasmic processes ("veils"). (ii) Langerhans cells in transition from epidermis to dermis were observed by transmission electron microscopy. Where Langerhans cells penetrated the basement membrane, the lamina densa was focally absent. (iii) This was highlighted by scanning electron microscopy, which presented the basement membrane as a tightly packed and dense network of fibrils. (iv) Scanning electron microscopy of the dermis revealed dendritic cells extending their cytoplasmic processes and clinging to collagen fibrils. (v) Entry of dendritic cells into dermal lymphatics was observed by transmission electron microscopy. It occurred by transmigration through intercellular spaces of adjacent endothelial cells. Entry through wide gaps between endothelial cells also seemed to take place. (vi) Dendritic cells inside the afferent lymphatics frequently carried material such as melanosomes and apoptotic bodies. These observations visualize the cumbersome pathway that dendritic cells have to take when they generate immunity.
Subject(s)
Langerhans Cells/physiology , Skin/cytology , Animals , Antigens/analysis , Basement Membrane/physiology , Cell Movement , Culture Techniques , Dendritic Cells/immunology , Dendritic Cells/physiology , Dermis/cytology , Epidermal Cells , Lymphatic System/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Microscopy, Electron , Microscopy, Electron, Scanning , Time FactorsABSTRACT
C-type lectin receptors help Langerhans cells (LC) to take up and process pathogens. Langerin/CD207 is a mannose-binding C-type lectin that is specifically expressed by LC. It is involved in antigen uptake in an as yet poorly defined way, and it is a major molecular constituent of Birbeck granules. We studied the emergence of Langerin expression in LC in epidermal sheets and cell suspensions during ontogeny. Langerin appears later than MHC II expression. Intracellular Langerin expression becomes apparent 2-3 d after birth. Only 10 days after birth all LC co-express Langerin. The intensity of Langerin expression reaches adult levels by 3 wk after birth. Early Langerin expression appears to correlate at least in part with the physical presence of Birbeck granules.
Subject(s)
Antigens, Surface/analysis , Epidermis/chemistry , Langerhans Cells/chemistry , Lectins, C-Type/analysis , Mannose-Binding Lectins/analysis , Age Factors , Animals , Flow Cytometry , Histocompatibility Antigens Class II/analysis , Mice , Mice, Inbred BALB CABSTRACT
Dendritic cells are professional antigen-presenting cells that initiate primary immunity. Migration from sites of antigen uptake to lymphoid organs is crucial for the generation of immune responses. We investigated the migratory pathways specifically of epidermal Langerhans cells by tracing them from the epidermis to the draining lymph nodes. This was possible with a new monoclonal antibody, directed against murine Langerin/CD207, a type II lectin specific for Langerhans cells. In situ, resident, and activated Langerhans cells express Langerin in the epidermis and on their way through dermal lymphatic vessels. Both emigrated and trypsinization-derived Langerhans cells expressed high levels of Langerin intracellularly but reduced it upon prolonged culture periods. Sizeable numbers of Langerin+ cells were found in skin draining lymph nodes but not in mesenteric nodes. Langerin+ cells localized to the T cells areas and rarely to B cell zones. Numbers of Langerin-expressing cells increased after application of a contact sensitizer. In the steady state, Langerhans cells in the skin-draining nodes expressed maturation markers, such as 2A1 and costimulatory molecules CD86 and CD40. These molecules, CD86 and CD40, were further upregulated upon inflammatory stimuli such as contact sensitization. Thus, the novel anti-Langerin monoclonal antibody permits the unequivocal visualization of migratory Langerhans cells in the lymph nodes for the first time and thereby allows to dissect the relative immunogenic or tolerogenic contributions of Langerhans cells and other types of dendritic cells.
Subject(s)
Antigens, Surface/immunology , Cell Movement/immunology , Epidermal Cells , Langerhans Cells/cytology , Lectins, C-Type/immunology , Lymph Nodes/cytology , Mannose-Binding Lectins , Animals , Antibodies, Monoclonal/pharmacology , Antibody Specificity , Antigens, Surface/analysis , Antigens, Surface/genetics , Cell Line , Cellular Senescence , Dermatitis, Contact/immunology , Dermatitis, Contact/pathology , Dermis/cytology , Fibroblasts/cytology , Immunophenotyping , Langerhans Cells/chemistry , Lectins, C-Type/analysis , Lectins, C-Type/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , T-Lymphocytes/cytology , TransfectionABSTRACT
HSV-1 is a very successful representative of the α-herpesvirus family, and â¼ 90% of the population is seropositive for this particular virus. Although the pathogen usually causes the well-known mild lesions on the lips, also, severe infections of the eye or the brain can be observed in rare cases. It is well known, that this virus can efficiently infect the most potent APCs, i.e., the DCs, in their immature and mature state. Although the infection of the iDC has been shown to be productive, infection of mMDDCs is believed to be abortive in the early phase of the viral replication cycle. In line with these findings, no virus particles can be detected in the supernatant of HSV-1-infected mMDDC. In this study, however, we show for the first time that this pathogen completes its replication cycle in mMDDCs. We detected the presence of viral gene transcripts of all three phases of the replication cycle, as well as of late viral proteins, and even the generation of small amounts of progeny virus. Although we could confirm the findings that these particles are not released into the supernatant, surprisingly, the newly generated viral particles can be passed on to Vero cells, as well as to primary keratinocytes in a cell-cell contact-dependent manner. Finally, we provide evidence that the viral gE is involved in the transfer of infectious virus from mMDDCs to other permissive cells.
Subject(s)
Dendritic Cells/immunology , Dendritic Cells/virology , Herpes Simplex/transmission , Herpesvirus 1, Human/physiology , Virion/physiology , Virus Replication , Animals , Blotting, Western , Cell Adhesion , Cell Communication , Cell Movement , Chlorocebus aethiops , Dendritic Cells/metabolism , Herpes Simplex/immunology , Herpes Simplex/metabolism , RNA, Messenger/genetics , RNA, Viral/genetics , Vero Cells , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
HIV-1 subverts antigen processing in dendritic cells (DCs) resulting in viral uptake, infection, and transfer to T cells. Although DCs bound monomeric gp120 and HIV-1 similarly, virus rarely colocalized with endolysosomal markers, unlike gp120, suggesting HIV-1 alters endolysosomal trafficking. Virus within DC intracellular compartments rapidly moved to DC-CD4+ lymphocyte synapses when introduced to CD4+ lymphocyte cultures. Although viral harboring and transfer from nonlysosomal compartments was transient, given DC-associated virus protein, nucleic acids, and infectious HIV-1 transfer to CD4+, lymphocytes decayed within 24 hours. However a second long-term transfer phase was apparent in immature DCs after 48 hours as a zidovudine-sensitive rise in proviral DNA. Therefore, DCs transfer HIV-1 to CD4+ lymphocytes in 2 distinct phases. Immature and mature DCs first divert virus from the endolysosomal pathway to the DC-T-cell synapse. Secondly, the later transfer phase from immature DCs is through de novo HIV-1 production. Thus, the controversy of DCs being infected or not infected for the mechanics of viral transfer to CD4+ lymphocytes can be addressed as a function of time.