Biomedical subjects

Daniel Hanau

Publications and source records attributed to Daniel Hanau.

At least 19 recordsLinked to original sources

PubMed2006

Reduced cytokine-mediated up-regulation of HLA-DR in TAP-deficient fibroblasts.

Human deficiency in transporter associated with antigen processing (TAP) is characterized by a very low surface expression of human leukocyte antigen (HLA) class I molecules in hematopoietic and non hematopoietic cells. Among the latter, TAP-deficient skin fibroblasts have previously been shown by us to be very sensitive to lysis by activated autologous NK cells, even in the presence of cytokines that up-regulate HLA class I expression, a mechanism sufficient to protect normal fibroblasts from NK cell-mediated killing. Our complementary investigations on two TAP-deficient skin fibroblast cell lines surprisingly revealed that in response to proinflammatory cytokines, up-regulation of HLA-DR molecules at the cell surface is much less marked than in the case of normal skin fibroblasts. In contrast, the surface molecules CD40 and CD54 increase as much as observed on normal cells, suggesting that TAP-deficient fibroblasts are able to efficiently transduce cytokine-mediated stimulating signals. Transfection of an intact TAP gene into one of the TAP-deficient fibroblast cell lines restored a normal HLA class I expression that strongly increased upon IFN-gamma-mediated stimulation, whereas HLA-DR still remained lower than in control cells. These results suggest that, in addition to the defect in the HLA class I antigen presentation pathway, HLA-DR up-regulation is affected in TAP-deficient skin fibroblasts through an unknown mechanism probably independent from TAP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

High expression of antioxidant proteins in dendritic cells: possible implications in atherosclerosis.

Dendritic cells (DCs) display the unique ability to activate naive T cells and to initiate primary T cell responses revealed in DC-T cell alloreactions. DCs frequently operate under stress conditions. Oxidative stress enhances the production of inflammatory cytokines by DCs. We performed a proteomic analysis to see which major changes occur, at the protein expression level, during DC differentiation and maturation. Comparative two-dimensional gel analysis of the monocyte, immature DC, and mature DC stages was performed. Manganese superoxide dismutase (Mn-SOD) reached 0.7% of the gel-displayed proteins at the mature DC stage. This important amount of Mn-SOD is a primary antioxidant defense system against superoxide radicals, but its product, H(2)O(2), is also deleterious for cells. Peroxiredoxin (Prx) enzymes play an important role in eliminating such peroxide. Prx1 expression level continuously increased during DC differentiation and maturation, whereas Prx6 continuously decreased, and Prx2 peaked at the immature DC stage. As a consequence, DCs were more resistant than monocytes to apoptosis induced by high amounts of oxidized low density lipoproteins containing toxic organic peroxides and hydrogen peroxide. Furthermore DC-stimulated T cells produced high levels of receptor activator of nuclear factor kappaB ligand, a chemotactic and survival factor for monocytes and DCs. This study provides insights into the original ability of DCs to express very high levels of antioxidant enzymes such as Mn-SOD and Prx1, to detoxify oxidized low density lipoproteins, and to induce high levels of receptor activator of nuclear factor kappaB ligand by the T cells they activate and further emphasizes the role that DCs might play in atherosclerosis, a pathology recognized as a chronic inflammatory disorder.

Adult↗

PubMed2006

Functional CD1a is stabilized by exogenous lipids.

Self-glycosphingolipids bind to surface CD1 molecules and are readily displaced by other CD1 ligands. This capacity to exchange antigens at the cell surface is not common to other antigen-presenting molecules and its physiological importance is unclear. Here we show that a large pool of cell-surface CD1a, but not CD1b molecules, is stabilized by exogenous lipids present in serum. Under serum deprivation CD1a molecules are altered and functionally inactive, as they are unable to present lipid antigens to T cells. Glycosphingolipids and phospholipids bind to, and restore functionality to CD1a without the contribution of newly synthesized and recycling CD1a molecules. The dependence of CD1a stability on exogenous lipids is not related to its intracellular traffic and rather to its antigen-binding pockets. These results indicate a functional dichotomy between CD1a and CD1b molecules and provide new information on how the lipid antigenic repertoire is immunologically sampled.

Antigens, CD1↗

PubMed2005

Assistance of microbial glycolipid antigen processing by CD1e.

Complexes between CD1 molecules and self or microbial glycolipids represent important immunogenic ligands for specific subsets of T cells. However, the function of one of the CD1 family members, CD1e, has yet to be determined. Here, we show that the mycobacterial antigens hexamannosylated phosphatidyl-myo-inositols (PIM6) stimulate CD1b-restricted T cells only after partial digestion of the oligomannose moiety by lysosomal alpha-mannosidase and that soluble CD1e is required for this processing. Furthermore, recombinant CD1e was able to bind glycolipids and assist in the digestion of PIM6. We propose that, through this form of glycolipid editing, CD1e helps expand the repertoire of glycolipidic T cell antigens to optimize antimicrobial immune responses.

Acylation↗

PubMed2005

The cellular pathway of CD1e in immature and maturing dendritic cells.

Dendritic cells (DCs) present antigens to T cells via CD1, HLA class I or class II molecules. During maturation, HLA class II-restricted presentation is optimized. The relocalization of CD1e from Golgi to endosomal compartments during DC maturation suggests also an optimization of the antigen-presentation pathway via CD1 molecules. We here detail the biosynthesis and cellular pathway of CD1e in immature and maturing DCs. Unlike the other CD1 molecules, CD1e was found to reach late endosomes through sorting endosomes, without passing through the plasma membrane in either immature or maturing cells. After induction of DC maturation, CD1e disappeared rapidly from the Golgi and was transiently localized in HLA-DR+ vesicles, while the number of CD1e+/CD1b+ compartments increased for at least 20 h. High-resolution light microscopy showed that, in immature DCs, CD1e+ vesicles were often in close apposition to EEA1+ or HLA-DR+ compartments, while CD1e displayed a nearly exclusive distribution in the lysosomes of mature DCs, a finding corroborated by immunoelectron microscopy. During maturation, CD1e synthesis progressively declined, while the endosomal cleavage of CD1e still occurred. Thus, CD1e displays peculiar properties, suggesting an unexpected role among the family of CD1 antigen-presenting molecules.

Antigens, CD1↗

PubMed2004

Dendritic cells cross-present HIV antigens from live as well as apoptotic infected CD4+ T lymphocytes.

A better understanding of the antigen presentation pathways that lead to CD8(+) T cell recognition of HIV epitopes in vivo is needed to achieve better immune control of HIV replication. Here, we show that cross-presentation of very small amounts of HIV proteins from apoptotic infected CD4(+) T lymphocytes by dendritic cells to CD8(+) T cells is much more efficient than other known HIV presentation pathways, i.e., direct presentation of infectious virus or cross-presentation of defective virus. Unexpectedly, dendritic cells also take up actively antigens into endosomes from live infected CD4(+) T lymphocytes and cross-present them as efficiently as antigens derived from apoptotic infected cells. Moreover, live infected CD4(+) T cells costimulate cross-presenting dendritic cells in the process. Therefore, dendritic cells can present very small amounts of viral proteins from infected T cells either after apoptosis, which is frequent during HIV infection, or not. Thus, if HIV expression is transiently induced while costimulation is enhanced (for instance after IL-2 and IFNalpha immune therapy), this HIV antigen presentation pathway could be exploited to eradicate latently infected reservoirs, which are poorly recognized by patients' immune systems.

Amino Acid Sequence↗

PubMed2004

HIV type 1-infected dendritic cells induce apoptotic death in infected and uninfected primary CD4 T lymphocytes.

In addition to their essential role in adaptive immunity, dendritic cells (DCs) participate in innate immunity. In the context of measles virus (MV) or cytomegalovirus infections, they develop cytotoxic functions that may contribute in vivo to the elimination of virus-infected cells, but that also kill infected and noninfected T lymphocytes. Because the human immunodeficiency virus (HIV) induces T cell depletion through mechanisms that are still obscure, we investigated its ability to trigger DC cytotoxicity. When incubated with HIV, monocyte-derived DCs induced apoptosis in MDA-231 cells, which are sensitive to MV-induced DC cytotoxicity, and in uninfected as well as HIV-infected H9 CD4+ T cell lines. This apoptosis was inhibited by a mixture of FasL, TRAIL, TNF-alpha, and TWEAK inhibitors. Indeed, HIV infection induced or enhanced sensitivity to TRAIL, TNF-alpha, and TWEAK in H9 cells. Moreover, dendritic cells incubated with HIV-1 BAL or a wildtype HIV-1 isolate induced apoptosis in autologous primary CD4+ T lymphocytes, infected or not with a wild-type HIV-1 isolate. Therefore, induction of DC cytotoxicity by HIV may be relevant to in vivo HIV infection. Induction of cytotoxicity in DCs by HIV might contribute to HIV-associated T cell depletion through induction of apoptosis, especially in the early stages of infection. It may also contribute to elimination of infected cells in vivo, thereby enhancing cross-presentation of HIV by DCs. Therefore this new cytotoxic function of DCs may play an important role in innate and adaptive immunity during HIV infection.

Apoptosis↗

PubMed2004

Modified vaccinia virus Ankara induces moderate activation of human dendritic cells.

Modified vaccinia virus Ankara (MVA) is a highly attenuated strain known to be an effective vaccine vector. Here it is demonstrated that MVA, unlike standard vaccinia virus (VACV) strains, activates monocyte-derived human dendritic cells (DCs) as testified by an increase in surface co-stimulatory molecules and the secretion of pro-inflammatory cytokines. Inhibition of virus gene expression by subjecting MVA to UV light or heat treatment did not alter its ability to activate DCs. On the other hand, standard VACV strains activated DCs if virus gene expression was prevented by prior UV light or heat treatment. These results suggest that MVA or standard VACV particles are responsible for DC activation but, in the case of standard VACV strains, virus gene expression prevents activation. Additional experiments showed that DCs were activated by MVA-infected HeLa cells and, under these conditions, could induce secretion of gamma interferon from T lymphocytes more efficiently than if a replication-competent VACV strain was employed. These data provide one explanation for the remarkable immune-stimulating capacity of MVA in the absence of virus multiplication.

Antigens, CD↗

PubMed2004

Reproduction of Langerin/CD207 traffic and Birbeck granule formation in a human cell line model.

Birbeck granules (BG) are organelles specific to Langerhans cells (LCs), which form where the C-type lectin Langerin accumulates. Their function remains obscure due to morphologic and dynamic alterations induced by maturation of isolated LC. In this study, we attempted to reconstitute Langerin traffic and BG formation in the endosomal pathway of a human melanoma cell line. In the selected Langerin-transfected cell line, M10-22E, Langerin is distributed between the early recycling endosomal compartment and the plasma membrane, as in LC. Whereas mainly concentrated in membranes related to the Rab11(+) endosomal recycling compartment at the steady state, Langerin also recycles in M10-22E cells and drives BG biogenesis in the endosomal recycling compartment. Interruption of endocytosis or recycling induces redistribution of intracellular Langerin with an associated alteration in BG location and morphology. We have, therefore, generated a stable, Langerin-transfected cell line in which Langerin traffic and distribution and BG morphology replicate that seen in freshly isolated LC. This practical model can now be used to further delineate the nature and function of BG.

Antigens, CD↗

PubMed2004

Human epidermal langerhans cells express the immunoregulatory enzyme indoleamine 2,3-dioxygenase.

Langerhans cells (LC) are a special subset of dendritic cells integrating cutaneous immunity. The study of LC function is of major interest not only for efforts of vaccine design and immunotherapy but also for gaining an insight into the pathogenesis of immune-mediated cutaneous diseases and neoplasias. Recently, defined antigen-presenting cells were described that express indoleamine 2,3-dioxygenase (IDO) and inhibit T cell proliferation in vitro and in vivo. Here, we show that stimulation with interferon-gamma (IFN-gamma) induces the expression of functionally active IDO in highly purified human epidermal LC. The induction of IDO after stimulation of LC with IFN-gamma seems to follow a defined kinetic with rapid upregulation followed by a downregulation after about 24 h of culture. Accordingly, proliferation of T cells induced by anti-CD3 antibodies was modulated by supernatants of IFN-gamma-activated human epidermal LC. Importantly, downregulation of T cell proliferation by supernatants of 24 h IFN-gamma-activated LC was prevented by inhibition of IDO. These results indicate that LC not only have the capacity to stimulate but also to inhibit T cells, and suggest that LC possess an immunoregulatory function in promoting T cell tolerance by production of IDO.

Antineoplastic Agents↗

PubMed2003

Common characteristics of the human and rhesus macaque CD1e molecules: conservation of biochemical and biological properties during primate evolution.

In humans, a family of five genes encodes the CD1 molecules. Four of these proteins, CD1a, b, c, and d, are expressed on the plasma membrane and traffic between the cell surface and endocytic compartments, where they are loaded with antigenic glycolipids. The existence of human CD1e was demonstrated recently. This molecule surprisingly remains inside the cell, accumulating mainly in the Golgi compartments of immature dendritic cells and in the late endosomes of mature dendritic cells. In the latter compartments, CD1e is cleaved and becomes soluble. To determine whether these properties were specific to human CD1e, we investigated the presence and characteristics of CD1e in the rhesus macaque, an evolutionarily distant species of the primate lineage. Our results show that the cellular and biochemical properties of the human and simian CD1e molecules are similar, suggesting that the particular intracellular distribution of CD1e is important for its physiological and/or immunological function.

Amino Acid Sequence↗

PubMed2003

Indoleamine 2,3-dioxygenase-expressing antigen-presenting cells and peripheral T-cell tolerance: another piece to the atopic puzzle?

There is growing evidence that dendritic cells, the major antigen-presenting cells and T-cell activators, have a broad effect on peripheral T-cell tolerance and regulation of immunity. Very recently, a new feature of regulatory antigen-presenting cells was observed. Certain dendritic cells, monocytes, and macrophages express the enzyme indoleamine 2,3-dioxygenase, and thus because of enhanced degradation of the essential amino acid tryptophan, they modulate T-cell activity in specific local tissue environments. In this review we discuss the various and apparently disparate effects of indoleamine 2,3-dioxygenase induction in cells of the immune system. We place current knowledge about this mechanism in the context of atopy. We introduce the hypothesis that tryptophan degradation might add to the ability to control and downregulate allergen-specific T-cell responses in atopic individuals.

Antigen-Presenting Cells↗

PubMed2003

Cored tubules are present in human epidermal Langerhans cells.

Cored tubules are ultrastructural organelles described to date only in murine cells belonging to the Langerhans cell family and located in the dermis and its draining lymph nodes. These organelles, the function of which is unknown, differ from Birbeck granules and are interestingly not found in murine epidermal Langerhans cells. In this work we demonstrate that cored tubules are present in freshly isolated human epidermal Langerhans cells. The tubules were found to be interconnected with structures known to belong to the early endosomal pathway and could be immunolabeled with gold-conjugated anti-CD1a and anti-Langerin monoclonal antibodies, but only at 37 degrees C. At this temperature such antibodies are able to progress from the early sorting endosomes to the early recycling endosomes, which in human Langerhans cells include the Birbeck granules. These findings strongly suggest that cored tubules form part of the early recycling compartment.

Antigens, CD↗

PubMed2003

Two human immunodeficiency virus vaccinal lipopeptides follow different cross-presentation pathways in human dendritic cells.

An efficient vaccine against human immunodeficiency virus (HIV) must induce good cellular immune responses. To do this, it must be processed and presented by dendritic cells, which are required for primary T-lymphocyte stimulation. We have previously shown that a model lipopeptide containing a short epitopic peptide from HIV-1 was endocytosed and presented in association with major histocompatibility complex class I molecules by human dendritic cells to specific CD8(+) T lymphocytes, but the cross-presentation pathway needed to be precisely determined. We have studied a longer lipopeptide (Pol(461-484)) and another lipopeptide (Nef(66-97)) currently being used in vaccine trials. Like the shorter lipopeptide, the rhodamine-labeled Pol(461-484) lipopeptide was internalized by endocytosis, as assessed by confocal microscopy. The lipopeptides were processed by dendritic cells and presented to CD8(+) T cells specific for the HLA-A*0201-restricted Pol(476-484) and the HLA-A*0301-restricted Nef(73-82) epitope, respectively. Presentation of both lipopeptides was inhibited by brefeldin A. Presentation of the Pol lipopeptide was inhibited by epoxomycin, a proteasome-specific inhibitor, but not by monensin. This shows that it gained access to the cytosol to be digested by the proteasome. In contrast, presentation of the Nef lipopeptide was not inhibited by epoxomycin but was inhibited by monensin, a classical inhibitor of acid-dependent endosomal enzyme activity, indicating an endocytic processing pathway yielding to major histocompatibility complex class I-restricted presentation. Therefore, the two lipopeptides followed different cross-presentation pathways, both resulting in efficient presentation to CD8(+) T lymphocytes.

AIDS Vaccines↗

PubMed2002

Kinetics of gene induction after FcepsilonRI ligation of atopic monocytes identified by suppression subtractive hybridization.

The high-affinity receptor for IgE, FcepsilonRI, on APCs plays an important role in the initiation and chronicity of inflammatory atopic diseases. To understand the molecular regulation of FcepsilonRI-mediated processes, differentially expressed genes are of great interest to be identified. Suppression subtractive cDNA hybridization has been used to identify genes induced after FcepsilonRI stimulation on atopic monocytes. Overexpression of the identified genes was determined by semiquantitative RT-PCR analysis of transcripts from the tester (stimulated) and driver (unstimulated) monocytes. Results were confirmed and kinetics of the transcripts established using blood cells from additional atopics at 4 and 24 h of FcepsilonRI induction. The following sequences were identified: monocyte chemoattractant protein 1, macrophage-inflammatory protein 1beta, IL-6, beta(A) subunit of inhibin/activin, IFN-stimulated gene of 54 kDa, IL-1R antagonist, and kynurenine 3-monooxygenase. Chemokines are highly expressed during the early and late phase after FcepsilonRI cross-linking, whereas proinflammatory and differentiation stimuli rapidly decline after an initial overexpression. Kynurenine 3-monooxygenase, an enzyme involved in the degradation of the amino acid tryptophan, is significantly up-regulated during the late phase after 24 h of FcepsilonRI induction. These results demonstrate that the analysis of the profile of gene induction following activation of FcepsilonRI on atopic monocytes may reveal how these cells might participate in the regulation of atopic disorders.

Base Sequence↗

PubMed2002

Evidence of a trimolecular complex involving LPS, LPS binding protein and soluble CD14 as an effector of LPS response.

The kinetics of the interaction of lipopolysaccharide (LPS), lipopolysaccharide binding protein (LBP) and CD14 was studied using surface plasmon resonance. The association and dissociation rate constants for the binding of LPS and rsCD14 were 2.9 x 10(4) M(-1) s(-1) and 0.07 s(-1) respectively, yielding a binding constant of 4.2 x 10(5) M(-1). Significantly, the presence of LBP increased not only the association rate but also the association constant for the interaction between LPS and CD14 by three orders of magnitude. Our experimental results suggest that LBP interacts with LPS and CD14 to form a stable trimolecular complex that has significant functional implications as it allows monocytes to detect the presence of LPS at a concentration as low as 10 pg/ml or 2 pM, and to respond by secreting interleukin-6. Thus, LBP is not merely transferring LPS to CD14 but it forms an integral part of the LPS-rLBP-rsCD14 complex.

Acute-Phase Proteins↗

PubMed2002

Flt3+ macrophage precursors commit sequentially to osteoclasts, dendritic cells and microglia.

BACKGROUND: Macrophages, osteoclasts, dendritic cells, and microglia are highly specialized cells that belong to the mononuclear phagocyte system. Functional and phenotypic heterogeneity within the mononuclear phagocyte system may reveal differentiation plasticity of a common progenitor, but developmental pathways leading to such diversity are still unclear. RESULTS: Mouse bone marrow cells were expanded in vitro in the presence of Flt3-ligand (FL), yielding high numbers of non-adherent cells exhibiting immature monocyte characteristics. Cells expanded for 6 days, 8 days, or 11 days (day 6-FL, day 8-FL, and day 11-FL cells, respectively) exhibited constitutive potential towards macrophage differentiation. In contrast, they showed time-dependent potential towards osteoclast, dendritic, and microglia differentiation that was detected in day 6-, day 8-, and day 11-FL cells, in response to M-CSF and receptor activator of NFkappaB ligand (RANKL), granulocyte-macrophage colony stimulating-factor (GM-CSF) and tumor necrosis factor-alpha (TNFalpha), and glial cell-conditioned medium (GCCM), respectively. Analysis of cell proliferation using the vital dye CFSE revealed homogenous growth in FL-stimulated cultures of bone marrow cells, demonstrating that changes in differential potential did not result from sequential outgrowth of specific precursors. CONCLUSIONS: We propose that macrophages, osteoclasts, dendritic cells, and microglia may arise from expansion of common progenitors undergoing sequential differentiation commitment. This study also emphasizes differentiation plasticity within the mononuclear phagocyte system. Furthermore, selective massive cell production, as shown here, would greatly facilitate investigation of the clinical potential of dendritic cells and microglia.

Animals↗

PubMed2002

FcepsilonRI induces the tryptophan degradation pathway involved in regulating T cell responses.

FcepsilonRI is suspected to play a pivotal role in the pathophysiology of atopic disorders such as atopic dermatitis. In search for genes differentially regulated by FcepsilonRI on APCs, a differential cDNA bank of receptor-stimulated and unstimulated monocytes was established. By means of suppression subtractive hybridization, we identified kynurenine 3-monooxygenase and subsequently indoleamine 2,3-dioxygenase (IDO) to be overexpressed in FcepsilonRI-activated monocytes. IDO is the rate-limiting enzyme in the catabolism of the essential amino acid tryptophan. We show that cross-linking of FcepsilonRI on monocytes results in low tryptophan concentrations associated with impaired T cell stimulatory capacity. Importantly, T cell suppression could be prevented by the addition of tryptophan or inhibition of IDO. Moreover, stimulation of T cells by FcepsilonRI-activated monocytes was increased compared with T cell stimulation by nonactivated monocytes if exogenous supply of tryptophan was available. We speculate that the expression of IDO by FcepsilonRI(+) APCs in vivo allows these cells to regulate T cell responses in atopic disorders by inhibiting or stimulating T cell proliferation, depending on the metabolic environment.

Antigen-Presenting Cells↗