Hella Stössel

Disruption of the langerin/CD207 Gene Abolishes Birbeck Granules without a Marked Loss of Langerhans Cell Function

ABSTRACT 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.

Two populations of splenic dendritic cells detected with M342, a new monoclonal to an intracellular antigen of interdigitating dendritic cells and some B lymphocytes

A monoclonal has been isolated that labels an intracellular antigen in dendritic cells and some B cells. The M342 hamster immunoglobulin was selected because it stained cells in the periarterial sheaths of spleen, the deep cortex of lymph node, and the thymic medulla –‐ the same regions in which one finds interdigitating cells, the presumptive in situ counterparts of isolated lymphoid dendritic cells. M342 labeled an antigen within granules of isolated dendritic cells, but only in cells that had been cultured for a day and not in fresh isolates. This extends recent findings that most freshly isolated spleen dendritic cells are located in the periphery of the white pulp nodule and may serve as precursors for the periarterial pool of interdigitating cells, the site for M342 staining in situ. By electron microscopic immunolabeling, the M342 antigen was found exclusively in a type of multivesicular body. M342 staining was not found in mononuclear phagocytes from blood and peritoneal cavity. Peritoneal B cells expressed M342+ granules, and upon appropriate stimulation splenic B cells developed reactive granules as well. We conclude that M342 is a strong marker for interdigitating cells. Its existence reveals intracellular specializations in the vacuolar system of antigen‐presenting cells including subsets of dendritic cells.

Resolution of de novo HIV production and trafficking in immature dendritic cells

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.

Endothelial cells from cord blood CD133+CD34+ progenitors share phenotypic, functional and gene expression profile similarities with lymphatics

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‐α, 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.

A Fusion Inhibitor Prevents Spread of Immunodeficiency Viruses, but Not Activation of Virus-Specific T Cells, by Dendritic Cells

ABSTRACT Dendritic cells (DCs) play a key role in innate immune responses, and their interactions with T cells are critical for the induction of adaptive immunity. However, immunodeficiency viruses are efficiently captured by DCs and can be transmitted to and amplified in CD4+ T cells, with potentially deleterious effects on the induction of immune responses. In DC-T-cell cocultures, contact with CD4+, not CD8+, T cells preferentially facilitated virus movement to and release at immature and mature DC-T-cell contact sites. This occurred within 5 min of DC-T-cell contact. While the fusion inhibitor T-1249 did not prevent virus capture by DCs or the release of viruses at the DC-T-cell contact points, it readily blocked virus transfer to and amplification in CD4+ T cells. Higher doses of T-1249 were needed to block the more robust replication driven by mature DCs. Virus accumulated in DCs within T-1249-treated cocultures but these DCs were actually less infectious than DCs isolated from untreated cocultures. Importantly, T-1249 did not interfere with the stimulation of virus-specific CD4+ and CD8+ T-cell responses when present during virus-loading of DCs or for the time of the DC-T-cell coculture. These results provide clues to identifying strategies to prevent DC-driven virus amplification in CD4+ T cells while maintaining virus-specific immunity, an objective critical in the development of microbicides and therapeutic vaccines.

Herpes simplex virus type I (HSV‐1) replicates in mature dendritic cells but can only be transferred in a cell–cell contact‐dependent manner

HSV‐1 is a very successful representative of the α‐herpesvirus family, and ∼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.

Isolation and characterization of CD133 + CD34 + VEGFR-2 + CD45 − fetal endothelial cells from human term placenta☆

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.

Human herpesvirus-8 infection of umbilical cord-blood-derived CD34+ stem cells enhances the immunostimulatory function of their dendritic cell progeny

Abstract:  CD34+ progenitor cells carrying human herpesvirus‐8, Kaposis sarcoma‐associated herpesvirus (HHV‐8/KSHV), have been described in the peripheral blood of AIDS patients suffering from Kaposis sarcoma (KS). In this study, we investigated the influence of HHV‐8 on the differentiation of CD34+ progenitor cells. Native CD34+ cells derived from cord blood could be infected by a laboratory strain of HHV‐8, as shown by immunofluorescence staining and polymerase chain reaction, but no significant initial maturation/differentiation effects were observed. In addition, these infected cells were differentiated into immature and mature dendritic cells (DCs) using cytokine induction with recombinant human granulocyte‐macrophage colony‐stimulating factor (rhGm‐CSF), recombinant human tumor necrosis factor (rhTNF‐α) and recombinant human stem cell factor (rhSCF). Double immunofluorescence and flow cytometry studies demonstrated that virus infection did not impair the development of immature and mature DC populations. Subsequently, the immunostimulating capacity of DC populations was tested in a mixed lymphocyte reaction using allogeneic T‐cells. The HHV‐8‐infected CD34+ progenitor cell‐derived mature DC population showed a significantly enhanced antigen‐presenting capacity, compared to non‐infected DCs, which was not observed with the immature DCs. This suggests stimulation of DC function by HHV‐8 infection. Because there are only a small percentage of HHV‐8‐positive DCs in the preparations and because it is not clear whether infection is abortive or productive to some extent, this seems to be most likely due to an indirect viral effect.

Global degranulation of rat mast cells stimulated with DNP-polystyrene

Mediator release was studied in rat peritoneal mast cells sensitized with a mouse monoclonal anti-DNP IgE antibody, and stimulated with DNP-ornithine covalently attached to radio-derivatized polystyrene petri dishes. Cells releasing serotonin at maximal rates were investigated by transmission electron microscopy. Generalized exocytosis of granules could be observed, suggesting non-directional release of mediators, and non-compartmentalized action of second messengers in mast cells stimulated with polystyrene-bound DNP. Stimulation of sensitized mast cells by DNP covalently bound to the rigid polystyrene surface is consistent with extrinsic mechanisms proposed for Fc(epsilon)RI receptor action, and suggests that internalization of Fc(epsilon)RI is not needed for triggering cell degranulation.

Migration of dendritic cells into the lymphatics: the Langerhans cell example

Dendritic cells, including Langerhans cells of the epidermis and the mucous membranes, are key leukocytes for the initiation of adaptive immune responses as well as for the maintenance of peripheral tolerance. In the former regard they may well play an important, but as yet unrecognized role in the pathogenesis of Behcets disease. Epidermal Langerhans cells may serve as a paradigm for their counterparts in the mucosae. These cells efficiently take up (microbial) antigens, they process them into immunogenic MHC–peptide complexes, and they transport this form of antigen to the lymph nodes via lymphatic vessels. Depending on the milieu where Langerhans cells have encountered antigen (inflammatory versus non-inflammatory/steady-state), they make T cells proliferate and acquire effector functions (immunity) or render them unresponsive or even delete them (tolerance), respectively. In addition, plasmacytoid dendritic cells, a recently characterized type of dendritic cells, may also directly trigger innate responses (e.g. by secretion of type I interferons in response to virus). Studying the pathways and the regulation of dendritic cell migration might help to unravel a possible involvement of dendritic cells in Behcets disease. We present observations on the physical obstacles that dendritic cells migrating in the skin have to overcome until they reach dermal lymphatic vessels. Furthermore, we show that migration is critically dependent on the function of matrix metalloproteinases, in particular MMP-2 and MMP-9. It becomes evident that Langerhans cells indeed carry antigens (including self antigens such as melanosomes or apoptotoic bodies or tumor antigens such as particular cytokeratins) through the lymphatics. Given these observations it may be worth studying Langerhans cells and dermal dendritic cells in Behcets disease.