Direct Ni2+ antigen formation on cultured human dendritic cells.
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The possible direct antigen formation of Ni2+ on antigen-presenting cells (APCs) was studied with cultured human dendritic cells (DCs) obtained from 10 subjects contact allergic to Ni2+ and six non-allergic control individuals. All contact allergic subjects showed a significantly increased peripheral blood mononuclear cell (PBMC) response in vitro to Ni2+. DCs were expanded from the plastic-adherent cell fraction of PBMCs by culturing with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 7 days to obtain immature DCs, and with the addition of monocyte-conditioned medium for another 4 days, for DC maturation. The DCs were pulsed for 20 min with Ni2+ (50 micrometers) in protein-free Hank's balanced salt solution (HBSS) and added to freshly prepared autologous responder PBMCs. With five allergic subjects, immature DCs pulsed with Ni2+ demonstrated a significant capacity to activate Ni2+-reactive lymphocytes. With the remaining five patients and the six controls no difference in lymphocyte proliferation was observed between Ni2+-pulsed and non-pulsed immature DCs. In contrast, with mature Ni2+-pulsed DCs from both 'positive responder' (n=4) and 'non-responder' (n=4) patients, there was a significantly stimulated PBMC proliferation, whereas with the controls (n=4) still no activation was observed. Our results indicate that direct formation of the antigenic determinant of Ni2+ on APCs is possible and that Ni2+ uptake and processing mechanisms may not play a major role. Differences in the ease of activation of Ni2+-reactive lymphocytes are discussed in terms of a possible heterogeneity in the availability of Ni2+-reactive groups presented on endogenous peptides bound in the antigen binding groove of human leucocyte antigen (HLA) class-II molecules. (+info)
Chemotactic response toward chemokines and its regulation by transforming growth factor-beta1 of murine bone marrow hematopoietic progenitor cell-derived different subset of dendritic cells.
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Dendritic cells (DCs) are highly specialized antigen-presenting cells that distribute widely in all organs. DCs initiate the primary immune response and activate naive T cells and B cells responsible for the acquired immunity. In this study, CCR7 mRNA was proved to be expressed in DCs and their precursors derived from murine bone marrow-derived hematopoietic progenitor cells (HPCs), whereas CCR1 mRNA was expressed in both CD11b-/dullCD11c+ and CD11b+hiCD11c+ DC precursors. CCR6 mRNA was not detected in any murine DC populations. In agreement with the chemokine receptor mRNA expression by each population in the DC differentiation pathway, SLC (also termed as MIP-3beta), one of the ligands for CCR7, strongly and selectively chemoattracted both CD11b-/dullCD11c+ and CD11b+hiCD11c+ DC precursors (days 6 to 7) and more mature DCs (days 13 to 14). We have recently found that transforming growth factor-beta1 (TGF-beta1), a cytokine that is essential for the appearance of Langerhans cells in the skin, polarizes murine HPCs to generate Langerhans-like cells through monocyte/macrophage differentiation pathway. We observed here that TGF-beta1 not only inhibited the expression of CCR7 in DCs and DC precursors derived from HPCs, but also inhibited the migration of these cells in response to SLC. This is the first report describing the chemokine and chemokine receptors responsible for murine DC migration and downregulation of DC migration by TGF-beta1. (+info)
The role of human T-lymphotropic virus type 1 (HTLV-1)-infected dendritic cells in the development of HTLV-1-associated myelopathy/tropical spastic paraparesis.
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The development of human T-lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is closely associated with the activation of T cells which are HTLV-1 specific but may cross-react with neural antigens (Ags). Immature dendritic cells (DCs), differentiated from normal donor monocytes by using recombinant granulocyte-macrophage colony-stimulating factor and recombinant interleukin-4, were pulsed with HTLV-1 in vitro. The pulsed DCs contained HTLV-1 proviral DNA and expressed HTLV-1 Gag Ag on their surface 6 days after infection. The DCs matured by lipopolysaccharides stimulated autologous CD4(+) T cells and CD8(+) T cells in a viral dose-dependent manner. However, the proliferation level of CD4(+) T cells was five- to sixfold higher than that of CD8(+) T cells. In contrast to virus-infected DCs, DCs pulsed with heat-inactivated virions activated only CD4(+) T cells. To clarify the role of DCs in HAM/TSP development, monocytes from patients were cultured for 4 days in the presence of the cytokines. The expression of CD86 Ag on DCs was higher and that of CD1a Ag was more down-regulated than in DCs generated from normal monocytes. DCs from two of five patients expressed HTLV-1 Gag Ag. Furthermore, both CD4(+) and CD8(+) T cells from the patients were greatly stimulated by contact with autologous DCs pulsed with inactivated viral Ag as well as HTLV-1-infected DCs. These results suggest that DCs are susceptible to HTLV-1 infection and that their cognate interaction with T cells may contribute to the development of HAM/TSP. (+info)
Susceptibility of bovine antigen-presenting cells to infection by bovine herpesvirus 1 and in vitro presentation to T cells: two independent events.
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The aim of the present study was to develop an in vitro system for presentation of bovine herpesvirus 1 (BHV-1) antigens to bovine T lymphocytes and to characterize the antigen-presenting cells (APC) which efficiently activate CD4(+) T cells. Two approaches were used to monitor the infection of APC by BHV-1 as follows: (i) detection of viral glycoproteins at the cell surface by immunofluorescence staining and (ii) detection of UL26 transcripts by reverse transcription-PCR. The monocytes were infected, while dendritic cells (DC) did not demonstrate any detectable viral expression. These data suggest that monocytes are one site of replication, while DC are not. The capacities of monocytes and DC to present BHV-1 viral antigens in vitro were compared. T lymphocytes (CD2(+) or CD4(+)) from BHV-1 immune cattle were stimulated in the presence of APC previously incubated with live or inactivated wild-type BHV-1. DC stimulated strong proliferation of Ag-specific T cells, while monocytes were poor stimulators of T-cell proliferation. When viral attachment to the surface of the APC was inhibited by virus pretreatment with soluble heparin, T-cell proliferation was dramatically decreased. Unexpectedly, incubation of DC and monocytes with the deletion mutant BHV-1 gD-/-, which displays impaired fusion capacity, resulted in strong activation of T lymphocytes by both APC types. Collectively, these results indicate that presentation of BHV-1 antigens to immune T cells is effective in the absence of productive infection and suggest that BHV-1 gD-/- mutant virus could be used to induce virus-specific immune responses in cattle. (+info)
Immortalized cell lines derived from mice lacking both type I and type II IFN receptors unify some functions of immature and mature dendritic cells.
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Cells with dendritic morphology obtained from several organs of mice lacking both type I and II IFN receptors were immortalized by a retrovirus and analysed for their phenotype and for their function to induce cognate immune responses in vitro and in vivo. Two cell lines called AG101 (skin) and AG116 (brain) were cloned and analysed in more detail. They constitutively expressed the cell surface markers CD45, CD11b, MHC class II, F4/80, N418, B7-2 and ICAM1 but were CD8- and B220-negative. Cells from both lines were capable of taking up ovalbumin (OVA). The processed protein was presented to the OVA-specific T cell hybridoma BO97.105 which responded specifically with the production of IL-2. AG101 and AG116 cells were able to induce a mixed lymphocyte reaction as shown by a 50-fold increase of IL-2 production over background. Naive T cells were stimulated by antigen-primed AG101 and AG116, resulting in a T cell proliferation which was 20-30 times over background, and in IL-2 production it was 10 times the background. The capacity of AG101 or AG116 cells to prime naive T cells was directly compared with freshly isolated and cultured cutaneous dendritic cells (DC) from 129 Sv/Ev mice (wtDC). After cognate T cell interaction, IL-6 (20-100-fold) and IL-12 p40 (100-1000-fold) were similarly up-regulated in either AG101, AG116 or mature wtDC. To analyse the capacity of the immortalized DC to induce antibodies in vivo, cell line AG116 was permanently infected with Borna disease virus (BDV) which is unable to replicate in adult mice. One hundred and twenty-nine Sv/Ev mice injected with different cell numbers of AG116 carrying BDV (but not control cells) produced antibodies against the viral BDVp40 and BDVp24 protein. Therefore, the cell lines AG101 and AG116 appear to unify some functions of immature and mature DC. They are able to pick up antigen and process it. In the absence of externally added cytokines, the antigen presented on AG101 or AG116 cells drives T cells with an efficiency similar to mature DC. The cloned cell lines may prove to be useful to study both immune response and replication of infectious agents in the absence of functional interferon receptors. (+info)
Polarised expression pattern of focal contact proteins in highly motile antigen presenting dendritic cells.
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Dendritic cells are professional antigen presenting cells that capture antigens and migrate to lymphoid tissues to elicit specific T cell responses. Here we used an in vitro differentiation system for generating highly motile dendritic cells from chicken bone marrow progenitors by employing the conditional v-Rel estrogen receptor (ER) fusion protein v-RelER. Molecular mechanisms of dendritic cell motility were investigated. Differentiation of v-relER progenitors into dendritic cells is associated with a reduction in cell-cell and cell-extracellular matrix interactions as cells acquire motility. We demonstrate that v-relER progenitors and dendritic cells express several adhesion receptors and components of adhesion complexes. Differentiation of v-relER cells was accompanied by downregulation of focal adhesion kinase (FAK), a key molecule of adhesion complexes, but ectopic FAK expression did not affect cell adhesion and motility. Interestingly, v-relER dendritic cells exhibit a polarised expression pattern of actin and vimentin, with actin being highly concentrated at the leading edge of the cells where lamellipodia are formed. FAK, paxillin and tyrosine phosphorylated proteins are found at both poles of the cell and colocalise with actin at the leading edge, while surface beta1 integrin is confined to the uropod at the rear. CD34(+ )stem cell-derived human dendritic cells also exhibited an elongated bipolar morphology, mode of migration and a polarised pattern of actin-vimentin expression similar to v-relER dendritic cells. (+info)
Calcium responses elicited in human T cells and dendritic cells by cell-cell interaction and soluble ligands.
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The interactions between a human CD4+ T cell clone and monocyte-derived human dendritic cells (DC) were analyzed with an imaging system. The first question addressed was the relationship between the formation of a contact zone and the triggering of a Ca2+ response in the T cells, in the presence or absence of antigen. Interaction of T cells with DC pulsed with the antigen led to the formation of a stable contact zone, followed by the appearance in the T cells of large and sustained Ca2+ oscillations. In the absence of antigen, contact zones formed normally and, surprisingly, Ca2+ responses were also observed, characterized by rare and small transients. Antigen-independent Ca2+ responses were not MHC restricted. The possible influence of Ca2+ responses in the DC on the efficiency of antigen presentation was then Investigated. In DC, Ca2+ responses can be elicited by a variety of stimuli: cell adhesion, platelet-activating factor, UTP and chemotactic molecules (formyl-Met-Leu-Pro, RANTES, MIP-1beta and SDF-1alpha). Importantly, Ca2+ responses were also induced in approximately 30% of DC as a result of their interaction with T cells. However, the efficiency of antigen presentation (as judged by the percentage of T cells presenting a Ca2+ response) was independent of the Ca2+ level in DC. Thus, imaging the interactions between human T cells and DC led us to observe two novel phenomena: DC-induced but antigen-independent Ca2+ responses in T cells and T cell-induced Ca2+ responses in DC. (+info)
P2Z/P2X7 receptor-dependent apoptosis of dendritic cells.
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Macrophages and thymocytes express P2Z/P2X7 nucleotide receptors that bind extracellular ATP. These receptors play a role in immune development and control of microbial infections, but their presence on dendritic cells has not been reported. We investigated whether extracellular ATP could trigger P2Z/P2X7 receptor-dependent apoptosis of dendritic cells. Apoptosis could be selectively triggered by tetrabasic ATP, since other purine/pyrimidine nucleotides were ineffective, and it was mimicked by the P2Z receptor agonist, benzoylbenzoyl ATP, and blocked by magnesium and the irreversible antagonist, oxidized ATP. RT-PCR analysis confirmed the mRNA expression of the P2Z/P2X7 receptor and the absence of P2X1. Caspase inhibitors and cycloheximide had only a partial effect on the apoptosis, suggesting that a caspase-independent mechanism may also be operative. Brief treatment with ATP led to an increase in the intracellular calcium concentration and permeabilization of the plasma membrane to Lucifer yellow, which diffused throughout the dendritic cell cytosol. Other small extracellular molecules may thus attain a similar intracellular distribution, perhaps activating endogenous proteases that contribute to initiation of apoptosis. (+info)