IL 4 Humain (A61772)

The aim of this study was to define the effects on antigen-presenting cells of the expression of HIV antigens from an attenuated poxvirus vector. We have analyzed the transcriptional changes in gene expression following infection of human immature monocyte-derived dendritic cells (DC) with recombinant modified vaccinia virus Ankara (MVA) expressing the genes encoding the gp120 and Gag-Pol-Nef antigens of HIV type 1 clade B (referred to as MVA-B) versus parental MVA infection. Using microarray technology and real-time reverse transcription-PCR, we demonstrated that the HIV proteins induced the expression of cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex (MHC) genes. Levels of mRNAs for interleukin-1, beta interferon, CCR8, and SCYA20 were higher after HIV antigen production. MVA-B infection also modulated the expression of antigen processing and presentation genes: the gene for MICA was upregulated, whereas those for HLA-DRA and HSPA5 were downregulated. Indeed, the increased expression of the gene for MICA, a glycoprotein related to major histocompatibility complex class I molecules, was shown to enhance the interaction between MVA-B-infected target cells and cytotoxic lymphocytes. The expression profiles of the genes for protein kinases such as JAK1 and IRAK2 were activated after HIV antigen expression. Several genes included in the JAK-STAT and mitogen-activated protein kinase signaling pathways were regulated after HIV antigen expression. Our findings provide the first gene signatures in DC of a candidate MVA-B vaccine expressing four HIV antigens and identified the biological roles of some of the regulatory genes, like that for MICA, which will help in the design of more effective MVA-derived vaccines.

Complex cellular functions within immunoinflammatory cascades are conducted by networks of interacting genes. In this study, we employed a network modeling approach to dissect and interpret global gene expression patterns in allergen-induced Th cell responses that underpin human atopic disease. We demonstrate that a subnet of interconnected genes enriched for Th2 and regulatory T cell-associated signatures plus many novel genes is hardwired into the atopic response and is a hallmark of atopy at the systems level. We show that activation of this subnet is stabilized via hyperconnected "hub" genes, the selective disruption of which can collapse the entire network in a comprehensive fashion. Finally, we investigated gene expression in different Th cell subsets and show that regulatory T cell- and Th2-associated signatures partition at different stages of Th memory cell differentiation. Moreover, we demonstrate the parallel presence of a core element of the Th2-associated gene signature in bystander naive cells, which can be reproduced by rIL-4. These findings indicate that network analysis provides significant additional insight into atopic mechanisms beyond that achievable with conventional microarray analyses, predicting functional interactions between novel genes and previously recognized members of the allergic cascade. This approach provides novel opportunities for design of therapeutic strategies that target entire networks of genes rather than individual effector molecules.

Activin A is a dimeric protein, member of the transforming growth factor (TGF)-beta family that plays a crucial role in wound repair and in fetal tolerance. Emerging evidence also proposes activin A as a key mediator in inflammation. This study reports that activin A induces the directional migration of immature myeloid dendritic cells (iDCs) through the activation of ALK4 and ActRIIA receptor chains. Conversely, activin A was not active on plasmacytoid dendritic cells (DCs) or mature myeloid DCs. iDC migration to activin A was phosphatidylinositol 3-kinase gamma-dependent, Bordetella pertussis toxin- and cycloheximide-sensitive, and was inhibited by M3, a viral-encoded chemokine-binding protein. In a real-time video microscopy-based migration assay, activin A induced polarization of iDCs, but not migration. These characteristics clearly differentiated the chemotactic activities of activin A from TGF-beta and classic chemokines. By the use of combined pharmacologic and low-density microarray analysis, it was possible to define that activin-A-induced migration depends on the selective and polarized release of 2 chemokines, namely CXC chemokine ligands 12 and 14. This study extends the proinflammatory role of activin A to DC recruitment and provides a cautionary message about the reliability of the in vitro chemotaxis assays in discriminating direct versus indirect chemotactic agonists.

Loss of tolerance toward commensal bacteria has been invoked as a mechanism for Crohn's disease. IL-10 is a key anti-inflammatory cytokine that plays a role in induction and maintenance of tolerance. The aim of this study is to determine IL-10 production in response to bacterial components in Crohn's disease patients, who were classified according to their phenotypes as stricturing, penetrating, or inflammatory. Peripheral blood was obtained from Crohn's disease patients and healthy controls. Cytokine production was measured in whole blood cultures, isolated CD4(+) cells, and monocyte-derived dendritic cells (MDDCs). Under unstimulated conditions, IL-10, but not IL-12, was down-regulated significantly in blood cultures of patients with severe phenotypes, compared with inflammatory, nonpenetrating, nonstricturing Crohn's disease patients. In response to LPS, IL-10 was up-regulated more significantly in patients with no fistulae or fibrosis. Study of IL-10 production by isolated cell subsets showed that DCs, but not CD4(+) T cells, from penetrating Crohn's disease produced significantly less IL-10 in response to LPS. Differences were not associated with the 1082A/G polymorphism in the IL-10 gene promoter. We show a defect in IL-10 production in whole blood cell cultures and MDDCs in patients with severe forms of Crohn's disease. This defect in IL-10 production by a group of Crohn's disease patients may represent a mechanism mediating more severe manifestations of the disease. We propose that treatment with IL-10 or IL-10-inducing therapies could be of particular benefit to these group of patients.

Atopic asthma pathogenesis is driven by the combined effects of airway inflammation generated during responses to viral infections and aeroallergens, and both these pathways are regulated by dendritic cells (DC) that differentiate locally from monocytic precursors. These DCs normally exhibit a sentinel phenotype characterized by active Ag sampling but attenuated presentation capability, which limits the intensity of local expression of adaptive immunity. How this tight control of airway DC functions is normally maintained, and why it breaks down in some atopics leading to immunopathological changes in airway tissues, is unknown. We postulated that signals from adjacent airway epithelial cells (AEC) contribute to regulation of local differentiation of DC. We tested this in a coculture model containing both cell types in a GM-CSF-IL-4-enriched cytokine milieu characteristic of the atopic asthmatic airway mucosa. We demonstrate that contact with AEC during DC differentiation up-regulates expression of the function-associated markers MHC class II, CD40, CD80, TLR3, and TLR4 on DCs with concomitant up-regulation of Ag uptake/processing. Moreover, the AEC-conditioned DCs displayed increased LPS responsiveness evidenced by higher production of IL-12, IL-6, IL-10, and TNF-alpha. The Th2 memory-activating properties of AEC-conditioned DCs were also selectively attenuated. Data from microarray and blocking experiments implicate AEC-derived type 1 IFNs and IL-6 in modulation of DC differentiation. Collectively, these findings suggest that resting AECs modulate local DC differentiation to optimize antimicrobial defenses in the airways and in the process down-modulate capacity for expression of potentially damaging Th2 immunity.

Plasmacytoid dendritic cells (pDC) are an important component of the innate immune response, producing large amounts of alpha interferon in response to viral stimulation in vitro. Under noninflammatory conditions, pDC are not found in the skin and are restricted in location to the blood and lymph nodes. Therefore, their role in mucosal and cutaneous herpes simplex virus (HSV) infection has not been well-defined. In this study we show a role for human pDC in the immune response to HSV infection. First, by confocal microscopy we showed that pDC infiltrate the dermis of recurrent genital herpes simplex lesions at early and late phases, often at the dermo-epidermal junction. We then showed that pDC in vitro are resistant to HSV infection despite expressing the entry receptors CD111, CD112, and HVE-A. Within the lesions, pDC were found closely associated with CD3(+) lymphocytes and NK cells, especially those which were activated (CD69(+)). Furthermore, these HSV-exposed pDC were able to stimulate virus-specific autologous T-lymphocyte proliferation. We conclude from this work that pDC may contribute to the immune control of recurrent herpes virus infection in vivo.

The death of dendritic cells (DCs) can potentially influence immune responses by affecting the duration of DC stimulation of lymphocytes. Here, we report that cultured mature monocyte-derived DCs manifest early mitochondrial damage (i.e. within 24 hrs), characterized by mitochondrial membrane potential (psi Delta m) disruption and mitochondrial release of pro-apoptotic factors, followed by reactive oxygen species (ROS) production and activation of caspases. Afterwards, DCs with mitochondrial alterations are condemned to undergo apoptosis and necrosis. Macroarray analysis results (validated by real time quantitative-PCR (QRT-PCR) and immunoblotting), showed up-regulation of the pro-apoptotic member of the Bcl-2 family, Bim, while expression of several anti-apoptotic molecules was down-regulated. Importantly, pre-apoptotic DCs (characterized by a low Delta psi m) showed a modified phenotype, with down-regulation of HLA-DR and of the co-stimulatory molecules CD80 and CD86. Moreover, sorted viable low psi Delta m DCs were unable to activate allogeneic T cells, indicating that pre-apoptotic DCs have already lost some of their immuno-stimulatory capabilities long before any detectable signs of death occur. Perturbations to mitochondrial respiration with rotenone identified the same modifications to DC immune functions. These data indicate a strong requirement for mitochondrial integrity for the immuno-stimulatory capacities of DC. Determining Delta psi m could be a useful parameter to select 'fully' functional DCs for anti-tumour vaccines.

Although recombinants based on the attenuated poxvirus vectors MVA and NYVAC are currently in clinical trials, the nature of the genes triggered by these vectors in antigen-presenting cells is poorly characterized. Using microarray technology and various analysis conditions, we compared specific changes in gene expression profiling following MVA and NYVAC infection of immature human monocyte-derived dendritic cells (MDDC). Microarray analysis was performed at 6 h postinfection, since these viruses induced extensive cytopathic effects, rRNA breakdown, and apoptosis at late times postinfection. MVA- and NYVAC-infected MDDC shared upregulation of 195 genes compared to uninfected cells: MVA specifically upregulated 359 genes, and NYVAC upregulated 165 genes. Microarray comparison of NYVAC and MVA infection revealed 544 genes with distinct expression patterns after poxvirus infection and 283 genes specifically upregulated after MVA infection. Both vectors upregulated genes for cytokines, cytokine receptors, chemokines, chemokine receptors, and molecules involved in antigen uptake and processing, including major histocompatibility complex genes. mRNA levels for interleukin 12beta (IL-12beta), beta interferon, and tumor necrosis factor alpha were higher after MVA infection than after NYVAC infection. The expression profiles of transcription factors such as NF-kappaB/Rel and STAT were regulated similarly by both viruses; in contrast, OASL, MDA5, and IRIG-I expression increased only during MVA infection. Type I interferon, IL-6, and Toll-like receptor pathways were specifically induced after MVA infection. Following MVA or NYVAC infection in MDDC, we found similarities as well as differences between these virus strains in the expression of cellular genes with immunological function, which should have an impact when these vectors are used as recombinant vaccines.

Mild heat stress can modulate the activities of immune cells, including dendritic cells (DC) and theoretically, would constitute an innovative approach capable of enhancing the antitumor functions of DC. Therefore, we tested the effects of mild heat stress on the physiology and viability of human monocyte-derived DC, the major type of DC used in tumor immunotherapy trials. We first designed a heat-stress protocol consisting of repetitive, sublethal heat shocks throughout the generation of DC. Using this protocol, we observed that heat stress did not perturb the morphology and the phenotype of immature or mature DC or the capacities of immature DC to uptake antigens efficiently. It is noteworthy that in response to heat stress, mature DC produced higher levels of IL-12p70 and TNF-alpha, which are two cytokines involved in the stimulation of inflammatory reaction, whereas IL-10 production remained low. After heat-stress exposure, mature DC have the full ability to stimulate naive T cells with Th1 response polarization (high IFN-gamma and low IL-4 production) in an allogeneic MLR. It is interesting that heat stress enhanced the migratory capacities of DC in response to MIP-3beta/CCL19. Finally, heat stress partly protected DC from apoptosis induced by cytokine withdrawal. Overall, these findings validate the feasibility of improving immune response by heating human monocyte-derived DC and provide a strong rationale for using mild heat stress in combination with DC vaccination to increase antitumor response.

Adaptive immune responses begin after productive immunosynaptic contacts formation established in secondary lymphoid organs by dendritic cells (DC) presenting the Ag to T lymphocytes. Despite its resemblance to the neurosynapse, the participation of soluble small nonpeptidic mediators in the intercellular cross-talk taking place during T cell-DC interactions remains poorly studied. In this study, we show that human DC undergoing maturation and in contact with T cells release significant amounts of glutamate, which is the main excitatory neurotransmitter in mammalians. The release of glutamate is nonvesicular and mediated by the DC-expressed Xc- cystine/glutamate antiporter. DC-derived glutamate stimulating the constitutively expressed metabotropic glutamate receptor 5 impairs T cell activation. However, after productive Ag presentation, metabotropic glutamate receptor 1 is expressed in T cells to mediate enhanced T cell proliferation and secretion of Th1 and proinflammatory cytokines. These data suggest that, during T cell-DC interaction, glutamate is a novel and highly effective regulator in the initiation of T cell-mediated immune responses.

Adenosine deaminase (ADA), a protein whose deficit leads to severe combined immunodeficiency, binds to the cell surface by means of either CD26, A(1) adenosine receptors, or A(2B) adenosine receptors. The physiological role of these interactions is not well understood. Our results show that by a 3-fold reduction in the EC(50) for the antigen, ADA potentiated T cell proliferation in autologous cocultures with antigen-pulsed immature or mature dendritic cells. Costimulation was not due to the enzymatic activity but to the interaction of ADA-CD26 complexes in T cells with an ADA-anchoring protein in dendritic cells. From colocalization studies, it is deduced that ADA colocalizing with adenosine receptors on dendritic cells interact with CD26 expressed on lymphocytes. This costimulatory signal in the immunological synapse leads to a marked increase (3- to 34-fold) in the production of the T helper 1 and proimmflamatory cytokines IFN-gamma, TNF-alpha, and IL-6.