Identification and characterization of equine blood plasmacytoid dendritic cells

Dendritic cells (DC) are antigen-presenting cells that can be classified into three major cell subsets: conventional DC1 (cDC1), cDC2 and plasmacytoid DCs (pDC), none of which have been identified in horses. Therefore, the objective of this study was to identify and characterize DC subsets in equine peripheral blood, emphasizing on pDC. Surface marker analysis allowed distinction of putative DC subsets, according to their differential expression of CADM-1 and MHC class II. Equine pDC were found to be Flt3⁺ CD4^low CD13⁻ CD14⁻ CD172a⁻ CADM-1⁻ MHCII^low. The weak expression of CD4 on equine pDC contrasts with findings in several other mammals. Furthermore, pDC purified by fluorescence-activated cell sorting were found to be the only cell subset able to produce large amounts of IFN-α upon TLR9-agonist stimulation. The pDC identity was confirmed by demonstrating high-levels of PLAC8, RUNX2 and TCF4 expression, showing pDC-restricted expression in other mammals.     

Identification and characterization of canine dendritic cells generated in vivo.

Emerging evidence suggests that host dendritic cells (DC) initiate and regulate graft-versus-host and graft-versus-tumor reactions after allogeneic hematopoietic cell transplantation (HCT). Even though decades of experimentation in the preclinical canine HCT model have substantially improved our understanding of the biology and safety of HCT in human patients, the in vivo phenotype of potent antigen-presenting cells in dogs is poorly defined. Therefore, peripheral blood leukocytes were obtained from dogs treated with recombinant human Flt3-ligand and phenotypically distinct cell populations, including putative DC, were purified by 4-color flow-cytometry and tested for their stimulatory potential in allogeneic mixed lymphocyte cultures (MLC). Cells characterized by surface expression of CD11c and HLA-DR, and absence of expression of CD14 and DM5, a marker of mature granulocytes, were found to be highly potent stimulators in allogeneic MLC. In contrast, all other immunophenotypically different cell populations tested had either weak or absent allostimulatory potential. Transmission electron microscopy of CD11c+/HLA-DR+/CD14-/DM5- cells revealed the morphology similar to that described for DC in humans and ex vivo-generated canine DC, including long cytoplasmic extensions, discrete lysosomes, and an abundant Golgi apparatus and endoplasmatic reticulum. In summary, CD11c+/HLA-DR+/CD14-/DM5- cells obtained from canine peripheral blood have functional and morphologic characteristics similar to those of human myeloid DC.     

Isolation and characterization of migratory human skin dendritic cells.

A method is described to isolate and characterize human skin dendritic cells (DC). This method is based on the migratory capacities of these cells. The cells migrated 'spontaneously' out of split-skin explants into the medium during a 24-h culture period and contained up to 75% CD1a+ cells. After removal of co-migrated T cells and macrophages, the highly enriched (> 95% CD1a+) DC showed potent allo-antigen-presenting capacities. About 25% of the CD1a+ cells were also positive for the dermal DC marker CD1b, whereas only 15-20% of the cells contained Birbeck granules, the characteristic cell organelle of the epidermal Langerhans cell. Before culture, CD1a+ DC were observed on cryostat sections not only in the epidermis but also in the dermis. After culture, the number of CD1a+ cells in both epidermis and dermis had decreased. Not all the cells had migrated during the culture period; some CD1a+ cells could still be detected in the epidermis and dermis after culture. Thus, using this method, potent allo-stimulating CD1a+ cells, migrating from both epidermis and dermis, can be obtained without the use of enzymes.     

Enrichment and characterization of dendritic cells from human bronchoalveolar lavages.

In the present study about 0.3% to 1.6% of human bronchoalveolar lavage (BAL) cells were identified as typical dendritic cells (DC), having an irregular outline, lobulated nucleus, and clear distinguishable acid phosphatase activity or EBM11 (anti-CD68) reactivity in a spot near the nucleus. After DC enrichment, using transient adherence to plastic, FcR-panning, and a density metrizamide gradient, a population containing 7-8% typical DC was obtained. This DC-enriched low density fraction, containing the highest percentages of DC, very strongly induced T cell proliferation in an allogeneic mixed leucocyte reaction (MLR), which was significantly higher than that induced by other partly (un)fractionated BAL cells. These data indicate that DC seem to be the major accessory cells in the BAL fluid, and therefore may be important in the regulation of T cell immune responses in the lung.     

食管黏膜树突状细胞表型分析

目的:研究食管黏膜树突状细胞(DCs)表型特点。方法:内镜下收集健康人食管黏膜标本,Ficoll-Hypaque密度梯度离心法分离食管黏膜标本中的单个核细胞,采用磁珠分选技术分离DCs,流式细胞仪分析食管黏膜DCs表型。结果:健康人食管黏膜含3类单个核细胞:HLA-DRhigh/CD13low,HLA-DRmed/CD13+和HLA-DR-/CD13+;HLA-DRhigh/CD13low细胞表达不同DCs亚群表面标志,是食管黏膜DCs;健康人食管黏膜DCsCD80、CD83、CD86表达水平低,是不成熟DCs。结论:成功分离了食管黏膜DCs,证实HLA-DRhigh/CD13low细胞是食管黏膜DCs。     

Functional and structural characterization of two populations of human monocyte-derived dendritic cells

BACKGROUND: The characterization of human monocyte-derived dendritic cells (HM-DC) subsets have been a very difficult and elusive task because of the lack of appropriate reagents. We, therefore utilized several diverse approaches to evaluate two populations of HM-DC including flow cytometry, ultra-structural evaluation by electron microscopy, and functional assays. In addition, we studied the kinetics of the expression of antigens on HM-DC at diverse intervals of time and identify surface markers and functional differences of these two HM-DC subsets. RESULTS: This study identified that a phenotype of HM-DC as defined by CD11c+, CD86+, and CD40+ could be separated in the presence or absence of TGF-beta1 into two different subsets of DC: (i) HM-DC without Birbeck granuli (Mo-DC) and (ii) HM-DC with Birbeck granuli (Mo-LC). Furthermore, the functional studies showed that the HM-DC treated with TGF-beta1 (Mo-LC) exhibited the presence of Birbeck granuli and could actively divide. In addition, after undergoing more than four cell divisions, these cells split into at least two additional subsets of Mo-LC: (iia) Mo-LC with high forward scatter (FSC) and (iib) Mo-LC with normal FSC. In contrast, the Mo-DC cultured in absence of TGF-beta1 did not exhibit Birbeck granuli, showed reduced ability to divide, and kept the normal FSC when analyzed. CONCLUSIONS: This study enabled us to determine in HM-DC: (i) the existence of antigenic and functional differences between various subpopulations of Mo-DC and Mo-LC; (ii) the existence of differences in the kinetics of antigens expression among the subsets of Mo-DC and Mo-LC; (iii) the existence of specific markers for each of the subpopulations of HM-DC.     

Identification and characterization of intestinal Peyer's patch interferon-alpha producing (plasmacytoid) dendritic cells

Recently, a subset of murine dendritic cells (DC) has been identified that resembles human plasmacytoid (pDC) the principal interferon-alpha (IFN-alpha) producing cells in blood. In this study, C57BL/10 (B10;H2b) mice were treated with fms-like tyrosine 3 kinase Ligand (Flt3L; 10 microg/d; i.p.; 10 days) that expands DC selectively in vivo. Putative pDC (CD11c+B220+) were identified in the subepithelial dome and in interfollicular regions of intestinal Peyer's patches (PP) from both normal and Flt3L-treated animals. Freshly-isolated, immunobead-purified CD11c+ DC from PP were flow-sorted to obtain lineage- (CD11b-CD19-) CD11c+ B220+ DC (purity>96%). Flow cytometric analysis revealed that these sorted PPpDC were negative for surface markers associated with myeloid DC (CD11b) and expressed only low levels of the "lymphoid-related" DC marker CD8alphaalpha+. They expressed low levels of costimulatory molecules and moderate MHC class II. They proved weak stimulators of na?ve allogeneic (C3H; H2k) T-cell proliferation. Cytospin preparations of sorted CD11c+B220+ cells revealed plasmacytoid morphology similar to that of human pDC. Immunocytochemistry and enzyme immunoassay revealed that, within 24-hour culture with Herpes simplex virus (10 p.f.u./cell), a subpopulation of stimulated (but not unstimulated) CD11c+B220+ DC produced and secreted IFN-alpha. This novel DC subset may play important roles in innate and adaptive immune responses of the gut and in the regulation of mucosal immune reactions.     

浆细胞样树突状细胞与疾病

目前对于浆细胞样树突状细胞(pDC)的来源、分布和功能研究不断深入,对于pDC在疾病发病、诊断、治疗中的作用正越来越受到关注。本文综述了pDC与抗病毒免疫特别是HIV感染、肿瘤、自身免疫病的关系,并总结了目前所发现的可以调控pDC生成和功能的药物,希望能为某些疾病的防治提供新的思路和方法。     

Isolation of follicular dendritic cells from human tonsils and adenoids. II. Immunocytochemical characterization

Follicular dendritic cells (FDC) are specialized cells found only within lymphoid follicles. They bind immune complexes and play a role in the presentation of antigen to follicular B cells and in the generation of B cell memory. In the present report the isolation of FDC from human tonsils and adenoids is described. These isolated cells have an unusual spherical arrangement and enclose lymphocytes within extensions of their membranes. Their ultrastructural features are similar to those observed in situ. The reactivity of isolated FDC with a number of monoclonal antibodies was analyzed by immunofluorescence and by immunostaining (at the electron microscopic level) with colloidal gold. In keeping with the results of previous investigations on tissue sections IgM, IgG and IgA (but not IgD) can be detected on the surface of isolated FDC, as can C3b receptors and the FDC-associated antigen detected by monoclonal antibody R4/23. The immunoglobulins associated with FDC are mostly embedded in an electron-dense material. The majority of the lymphoid cells enclosed within the membrane extensions of FDC are of B cell type. These results suggest that isolated FDC may be suitable for further in vitro investigation of their role in the humoral immune response.     

Immunophenotypic and functional characterization of cord blood dendritic cells

Dendritic cells (DCs) play a pivotal role in the activation of T cells, which are effector cells in graft-versus-host disease (GVHD). A low incidence of GVHD following cord blood (CB) transplantation has long been reported; despite this, little information is currently available on the characteristics of CB DCs. The goal of the present study was to investigate the immunophenotypic characteristics and distribution of CB DCs and their subsets. For that purpose we have analyzed 15 CB samples as compared to normal peripheral blood (PB) (n = 7) and blood from patients submitted to an allogeneic PB stem cell transplantation (allo-PBSCT) (n = 6). Our results show an overall decreased frequency of DCs in CB due to the presence of significantly lower numbers of CD123inter./CD33inter./CD16+ DCs. Phenotypically, CB DCs displayed a tendency to express lower levels of the gamma-chain interleukin-2 (IL-2) receptor (CD132) and of the CD86 co-stimulatory molecule, supporting a higher degree of immaturity for CB as compared to PB DCs. After activation of CB DCs with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) higher frequencies of cytokine-producing cells were found among CD123inter./CD33inter./CD16+ and CD123dim/CD33bright/CD16- DCs; however, when only the cytokine-producing DCs were considered, a significant decrease in the amount of different cytokine (e.g., IL-1beta and IL-6) produced per cell was observed especially for CD16+ CB DCs. These findings support a higher degree of immaturity for CB as compared to PB DCs that might contribute to explain, at least in part, the low incidence and severity of GVHD observed after CB transplantation.     

Identification and characterization of human thymic cortical dendritic macrophages that may act as professional scavengers of apoptotic thymocytes

We identify and characterize a special type of macrophage in the human thymic cortex that may act as professional scavengers of apoptotic thymocytes. These are large cells with clear cytoplasm, evenly distributed exclusively in the thymic cortex, and usually contain degraded nuclei in their cytoplasm. They are distinct from ordinary macrophages (OM) in the thymic cortex in expressing fascin, an actin-bundling protein specific for dendritic cells (DC), and in lacking lysozyme (LZM) and CD68. They are also different from DC in lacking major histocompatibility complex (MHC)-class II molecules. To distinguish them from OM and DC, we called them thymic cortical dendritic macrophages (TCDM). Both TCDM and OM are positive for DC-SIGN (CD209) and HAM56, whereas fascin(hi) MHC-class II(hi) medullary DC (mDC) are negative for these antigens. TCDM exhibit either dendritic or plump feature depending on cases examined. Plump TCDM usually contain several degraded nuclei, while dendritic TCDM contain one or two. These degraded nuclei are positive for active caspase-3 (aCasp-3), indicating that they are apoptotic thymocytes. In contrast to TCDM, LZM(hi) CD68(hi) OM are smaller round cells, distributed unevenly throughout the thymus, and do not contain apoptotic thymocytes at all. TCDM tend to adhere to capillaries with their dendrites or they make extensive contacts covering a large portion of the capillaries. Electron microscopic analysis confirmed the extensive contact between TCDM and capillaries and indicated that TCDM possess extremely electron-lucent, abundant cytoplasm with numerous tubulovesicular structures and secondary lysosomes. The finding of numerous condensed nuclei in most of the TCDM indicates that these cells represent a special type of fixed macrophages in the human thymic cortex, and that they play a central role in the clearance of apoptotic thymocytes.     

Identification and characterization of an ascorbic acid transporter in human granulosa-lutein cells.

Ascorbic acid serves a vital role as a pre-eminent antioxidant. In animals, it has been shown to be concentrated in granulosa and theca cells of the follicle, in luteal cells of the corpus luteum, and in the peripheral cytoplasm of the oocyte. We have previously identified hormonally-regulated ascorbic acid transporters in rat granulosa and luteal cells, and herein present preliminary evidence for the presence of a transporter for ascorbic acid in human granulosa-lutein cells. Granulosa-lutein cells were obtained from the follicular fluid of patients undergoing in-vitro fertilization. Following an overnight incubation, the cells were incubated with [14C]-ascorbic acid (0.15 microCi; 150 microM) and ascorbic acid uptake was determined. The uptake of ascorbic acid was saturable with a Michaeli's constant (Km) and maximum velocity (Vmax) of 21 microM and 3 pmol/10(6) cells/min respectively. Ouabain, low Na+ medium, and dinitrophenol significantly inhibited ascorbic acid uptake (P<0.05). Neither the presence of insulin, human chorionic gonadotrophin (HCG), insulin-like growth factor (IGF)-I, nor IGF-II affected the uptake of ascorbic acid in a statistically significant fashion. Following saturation of cellular uptake, the ascorbic acid level was estimated to be 1.04 pmoles/10(6) cells or approximately 1 mM, a high concentration similar to that seen in rat luteal cells. Active ascorbic acid transport in human granulosa-lutein cells appears to occur via a Na+ - and energy-dependent transporter, with high levels of ascorbic acid being accumulated in these cells.     

Origin and filiation of human plasmacytoid dendritic cells

Human plasmacytoid dendritic cells represent a rare population of leukocytes which produce high amounts of type I interferon in response to certain viruses. Although those cells were first described in 1958, there are still unsolved issues related to their origin and function. Recently, a leukemic counterpart of plasmacytoid dendritic cells was identified. Molecular approaches using either normal or leukemic plasmacytoid dendritic cells provide some new insights into the controversial lymphoid origin of those cells. The need for specific markers is still a critical aspect for the identification of plasmacytoid dendritic cells, whatever stage of differentiation, in normal as well as in pathological conditions. Hopefully, novel markers will allow delineation of the relationships between dendritic cells at different stages of differentiation/maturation along the myeloid and lymphoid lineages.     

Distribution of human colonic dendritic cells and macrophages

To define the phenotype of intestinal dendritic cells and macrophages, resected colonic specimens were used to obtain lamina propria cell suspensions by EDTA treatment, then enzymatic digestion. The phenotype of dendritic cell-enriched suspensions was compared with that of macrophage-enriched populations by immunocytochemistry using the avidin-biotin-peroxidase (ABC) system and immunoelectron microscopy. Dendritic cells expressed HLA-DR (L243) and HLA-DQ-associated (RFD1) antigens and CD68 in a perinuclear distribution. Staining for S100 was weak or absent. Macrophages also expressed HLA markers (L243 and RFD1) and CD68. The 25F9 antigen was expressed strongly, whilst CD14 was absent from cells isolated from non-inflamed tissues. To determine their anatomic distribution, immunohistochemistry was performed using single- and double-labelling techniques (ABC ± alkaline phosphatase anti-alkaline phosphatase method). Mutually exclusive subsets of 25F9⁺ and S100⁺cells were seen: 25F9⁺ macrophages were concentrated in a band immediately beneath the luminal epithelium; S100⁺/HLA-DR⁺ dendritic cells formed a reticular network throughout the lamina propria and beneath the basement membrane of the crypts. This distribution suggests that macrophages may help regulate intestinal responses by acting as the first line of defence against the entry of luminal antigens. A breach of the macrophage ‘barrier’ by invading antigens may necessitate the recruitment of T cell responses by immunostimulatory dendritic cells.     

The interactions between human dendritic cells and microbes; possible clinical applications of dendritic cells

The dendritic cells comprise several subsets that induce and regulate the immune responses against foreign and self-antigens, and that can therefore function as initiators of protective immunity and inducers of central or peripheral tolerance. The different subpopulations of dendritic cells interact with and also influence other cell populations of the immune system, such as T and B lymphocytes and natural killer cells. The factors that determine the given dendritic cell functions depend on the state of maturation and the local microenvironment. The interactions between dendritic cells and microorganisms are rather complex, but progress in the past few years has shed light on several aspects of these interactions. This review lays emphasis on the interactions between human dendritic cells, important components of the intima of arterial specimens at areas predisposed to atherosclerotic lesions, and Chlamydia pneumoniae and cytomegalovirus, the human pathogens most strongly implicated in the development of atherosclerosis. In addition, several examples of the potential clinical applications of dendritic cells are described.Received 13 January 2004; accepted by A. Falus 22 March 2004     

Generation and functional characterization of mouse monocyte-derived dendritic cells.

Dendritic cells (DC) are potent antigen-presenting cells with the unique capacity to initiate primary immune responses. As a result, DC are currently used in clinical studies to induce immunity against infectious disease and malignant cells. However, multiple DC subsets exist and it has been suggested that the type of DC may affect the immune response induced. The vast majority of DC used in experimental mouse tumor models is derived from bone marrow progenitors. In contrast, most in vitro as well as in vivo human studies involve the use of DC generated from adherent peripheral blood-derived monocytes in the presence of GM-CSF and IL-4. In the current report, we describe for the first time the generation and characterization of mouse monocyte-derived DC (MODC). The results indicate that mouse MODC display similar morphology, phenotype and immunostimulatory activity as compared to bone marrow-derived DC. Both DC subsets were able to efficiently take up and subsequently cross-present protein antigen to cytotoxic T cells. Moreover, we demonstrate that vaccination with peptide-loaded MODC mediates induction of tumor-reactive immunity in vivo. The isolation and characterization of mouse MODC will provide a valuable research tool to investigate fundamental aspects of DC biology and which DC subsets are most suitable to induce anti-tumor immunity.     

Recognition of autologous dendritic cells by human NK cells

NK cells can recognize and kill tumor as well as certain normal cells. The outcome of the NK-target interaction is determined by a balance of positive and negative signals initiated by different target cell ligands. We have previously shown that human NK cells kill CD40-transfected tumor targets efficiently, but the physiological significance of this is unclear. We now demonstrate that human NK cells can kill dendritic cells (DC), known to express CD40 and other co-stimulatory molecules. The killing was observed with polyclonal NK cells cultured short term in IL-2 as well as with NK cell clones as effectors, and with allogeneic as well as autologous DC as targets. NK cell recognition could be inhibited, but only partially, by preincubation of target cells with monoclonal antibodies against CD40, suggesting that this molecule may be one of several ligands involved. Addition of TNF-alpha of the cultures stimulated the development of a more mature DC phenotype, while addition of IL-10 resulted in a less mature phenotype, with lower expression of CD40 and other co-stimulatory molecules. Nevertheless, such DC were more NK susceptible than the differentiated DC. This may be partly explained by a reduced MHC class I expression observed on such cells, since blocking of MHC class I molecules on differentiated DC or CD94 receptors of NK cells led to increased NK susceptibility. The results show that NK cells may interact with DC, and suggest that the outcome of such interactions depend on the cytokine milieu.