Human Immature Dendritic Cells Research Articles

Inhibition of PGE2 and LTB4 production by dendritic cells induced by Paracoccidioides brasiliensis is associated with the non maturation of these cells

Event Abstract Back to Event Inhibition of PGE2 and LTB4 production by dendritic cells induced by Paracoccidioides brasiliensis is associated with the non maturation of these cells Reginaldo K. Fernandes1*, Tatiana F. Bachiega1, Daniela R. Rodrigues1, Márjorie A. Golim2, Luciane A. Dias-Melicio3, Helanderson A. Balderramas1, Ramon Kaneno4 and Ângela M. Soares1* 1 Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP) - Câmpus de Botucatu, Microbiologia e Imunologia - Instituto de Biociências, Brazil 2 Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Hemocentro, Brazil 3 Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Depto. Patologia - FMB - Botucatu, Brazil 4 Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP), Microbiologia e Imunologia, Brazil The fungus Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis, a systemic mycosis endemic in Latin America. Dendritic cells (DCs) play a crucial role in the detection of pathogens, triggering an initial response from the host as well as the instruction of the adaptive immune response. Mediators released by DCs, by an autocrine way, modulate their functions. Among these, we highlight the prostaglandins and leukotrienes. We therefore assessed whether human DCs produce PGE2 and LTB4 in response to fungus, the participation of recognition receptors of molecular patterns (PRRs) in this production, as well as the modulating role of these eicosanoids on phenotypic maturation of these cells. Human immature DCs (CD14-/ CD1ahigh/CD83low) derived from the differentiation of monocytes cultured with GM-CSF and IL-4 (7 days) released substantial concentrations of PGE2 and LTB4 which increased significantly in the case of PGE2, with incubation with LPS for 1h, 2h, 4h, 8h, 12h, 18h, 24h or 48h. However, the levels of PGE2 and LTB4 were significantly inhibited after challenge with two different strains of the fungus. Assays using monoclonal antibodies to block PRRS showed the involvement of mannose receptor in PGE2 inhibition by the fungus. Additionally, phenotyping assays showed that after challenge with the fungus, DCs did not change their phenotype of immature cells to mature ones. Accordingly, we detected a clear association between inhibition of the production of PGE2 and LTB4 fungus and inability to induce maturation of DCs. Assays using exogenous PGE2 and LTB4 confirmed this association. Acknowledgements Financial Support: FAPESP 2011/12477-0 / 2011/04556-8 Keywords: Eicosanoids, Dendritic Cells, prostaglandin, leukotriene, Paracoccidioides brasiliensis Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Fernandes RK, Bachiega TF, Rodrigues DR, Golim MA, Dias-Melicio LA, Balderramas HA, Kaneno R and Soares ÂM (2013). Inhibition of PGE2 and LTB4 production by dendritic cells induced by Paracoccidioides brasiliensis is associated with the non maturation of these cells. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00536 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 25 Apr 2013; Published Online: 22 Aug 2013. * Correspondence: Mr. Reginaldo K Fernandes, Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP) - Câmpus de Botucatu, Microbiologia e Imunologia - Instituto de Biociências, Botucatu-SP, Brazil, regiskeller@msn.com Prof. Ângela M Soares, Universidade Estadual Paulista "Júlio de Mesquita Filho" (UNESP) - Câmpus de Botucatu, Microbiologia e Imunologia - Instituto de Biociências, Botucatu-SP, Brazil, acsoares@ibb.unesp.br Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Reginaldo K Fernandes Tatiana F Bachiega Daniela R Rodrigues Márjorie A Golim Luciane A Dias-Melicio Helanderson A Balderramas Ramon Kaneno Ângela M Soares Google Reginaldo K Fernandes Tatiana F Bachiega Daniela R Rodrigues Márjorie A Golim Luciane A Dias-Melicio Helanderson A Balderramas Ramon Kaneno Ângela M Soares Google Scholar Reginaldo K Fernandes Tatiana F Bachiega Daniela R Rodrigues Márjorie A Golim Luciane A Dias-Melicio Helanderson A Balderramas Ramon Kaneno Ângela M Soares PubMed Reginaldo K Fernandes Tatiana F Bachiega Daniela R Rodrigues Márjorie A Golim Luciane A Dias-Melicio Helanderson A Balderramas Ramon Kaneno Ângela M Soares Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Reduced in vitro T‐cell responses induced by glutaraldehyde‐modified allergen extracts are caused mainly by retarded internalization of dendritic cells

Although allergen-specific immunotherapy is a clinically effective therapy for IgE-mediated allergic diseases, the risk of IgE-mediated adverse effects still exists. For this reason, chemically modified allergoids have been introduced, which may destroy IgE-binding sites while T-cell activation should be retained. The aim of the study was to analyse the differences between intact allergens and differently modified/aggregated allergoids concerning their internalization as well as T-cell and basophil activation. For this purpose human monocyte-derived immature dendritic cells (DC) were incubated with Phleum pratense or Betula verrucosa pollen extract or with the corresponding allergoids, modified with formaldehyde or glutaraldehyde. After an additional maturation process, the antigen-loaded mature DC were co-cultured with autologous CD4(+) T cells. Allergenicity was tested by leukotriene release from basophils. In addition, the uptake of intact allergens and allergoids by immature DC was analysed. The proliferation of, as well as the interleukin-4 (IL-4), IL-10, IL-13 and interferon-γ production by, CD4(+) T cells which had been stimulated with glutaraldehyde allergoid-treated DC was reduced compared with CD4(+) T cells stimulated with intact allergen-treated or formaldehyde allergoid-treated DC. In line with this, glutaraldehyde-modified allergoids were more aggregated and were internalized more slowly. Furthermore, only the allergoids modified with glutaraldehyde induced a decreased leukotriene release by activated basophils. These findings suggest that IgE-reactive epitopes were destroyed more efficiently by modification with glutaraldehyde than with formaldehyde under the conditions chosen for these investigations. Glutaraldehyde-modified allergoids also displayed lower T-cell stimulatory capacity, which is mainly the result of greater modification/aggregation and diminished uptake by DC.

Abstract SY40-02: Interplay between microenvironment and cancers

Abstract Tumors are highly adept at evading the immune system through a multitude of mechanisms, including the secretion of factors that modulate both innate and adaptive immunity. Matrix metalloproteinase-2 (MMP-2) is a proteolytic enzyme that degrades the extracellular matrix and is over expressed by many tumors, including melanoma. We recently documented the presence of MMP-2-specific CD4(+) T cells in tumor-infiltrating lymphocytes (TILs) in several melanoma patients (Cancer Cell 19:333, 2011). Strikingly, MMP-2-specific CD4(+) T cells displayed an inflammatory T(H)2 profile, mainly TNF-α, IL-4, and IL-13 and expressing GATA-3. When exposed to MMP-2, immature human dendritic cells (DCs) primed naïve CD4(+) T cells to differentiate into an inflammatory T(H)2 phenotype through OX40L expression and inhibition of IL-12p70 production. MMP-2 was subsequently found to degrade the type I IFN receptor, thereby preventing STAT1 phosphorylation, which is necessary for IL-12p35 production, and the subsequent formation of bioactive IL-12. More recently we have explored the mechanisms underlying the up regulation of OX40L and have identified signaling pathways and potential receptors that lead to this modulation. Active MMP-2, therefore, acts as an endogenous type 2 “conditioner” providing an explanation for the observed prevalence of detrimental type 2 responses in melanoma. Novel properties of MMP-2 on other components of the immune system, and on tumor progression, will be discussed along with DC-based strategies to alleviate immune suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY40-02. doi:1538-7445.AM2012-SY40-02

Strong CD8 + T cell antigenicity and immunogenicity of large foreign proteins incorporated in HIV-1 VLPs able to induce a Nef-dependent activation/maturation of dendritic cells

Virus-like particles (VLPs) are excellent tools for vaccines against pathogens and tumors. They can accommodate foreign polypeptides whose incorporation efficiency and immunogenicity however decrease strongly with the increase of their size. We recently described the CD8 + T cell immune response against a small foreign antigen (i.e., the 98 amino acid long human papilloma virus E7 protein) incorporated in human immunodeficiency virus (HIV)-1 based VLPs as product of fusion with an HIV-1 Nef mutant (Nef mut). Here, we extended our previous investigations by testing the antigenic/immunogenic properties of Nef mut-based VLPs incorporating much larger heterologous products, i.e., human hepatitis C virus (HCV) NS3 and influenza virus NP proteins, which are composed of 630 and 498 amino acids, respectively. We observed a remarkable cross-presentation of HCV NS3 in dendritic cells challenged with Nef mut–NS3 VLPs, as detected using a NS3 specific CD8 + T cell clone as well as PBMCs from HCV infected patients. On the other hand, when injected in mice, Nef mut–NP VLPs elicited strong anti-NP CD8 + T cell and CTL immune responses. In addition, we revealed the ability of Nef mut incorporated in VLPs to activate and mature primary human immature dendritic cells (iDCs). This phenomenon correlated with the activation of Src tyrosine kinase-related intracellular signaling, and can be transmitted from VLP-challenged to bystander iDCs. Overall, these results prove that Nef mut-based VLPs represent a rather flexible platform for the design of innovative CD8 + T cell vaccines.

Haemoglobin triggers chemotaxis of human monocyte-derived dendritic cells: Possible role in atherosclerotic lesion instability

ObjectiveMechanisms that drive innate immune cell recruitment into atherosclerotic lesions are still not well defined. We tested the role of haemoglobin (Hb) to promote chemotaxis, adhesion to endothelial cells and transendothelial migration of human monocytes and monocyte-derived immature dendritic cells (iDCs) and its possible role in atherogenic cell recruitment. Methods and resultsWe demonstrated that Hb triggers chemotaxis, adhesion to endothelial cells and transendothelial migration of monocytes and monocyte-derived iDCs. Innate immune cell chemotaxis significantly increased in the presence of Hb in a dose-dependent manner involving extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) activation and actin remodeling. The pre-treatment of cells with pre-titrated concentration of the anti-CD163 blocking antibody reduced the Hb-induced cell migration, thus suggesting the involvement of CD163 receptor. Conversely, N-acetyl cysteine and soluble Hb-scavenger protein haptoglobin (Hp) inhibited the Hb-induced iDC migration. Finally, spontaneous iDC migration significantly increased in the presence of serum of patients with haemorrhagic complicated plaques and partially decreased in the presence of Hp. ConclusionHb by interacting with CD163 on monocytes and iDCs might induce cell recruitment and activation within vascular wall, thus contributing to the complex cross talk of chemotactic signals that mediate atherosclerotic lesions instability.

The temporal dynamics of differential gene expression in Aspergillus fumigatus interacting with human immature dendritic cells in vitro.

Dendritic cells (DC) are the most important antigen presenting cells and play a pivotal role in host immunity to infectious agents by acting as a bridge between the innate and adaptive immune systems. Monocyte-derived immature DCs (iDC) were infected with viable resting conidia of Aspergillus fumigatus (Af293) for 12 hours at an MOI of 5; cells were sampled every three hours. RNA was extracted from both organisms at each time point and hybridised to microarrays. iDC cell death increased at 6 h in the presence of A. fumigatus which coincided with fungal germ tube emergence; >80% of conidia were associated with iDC. Over the time course A. fumigatus differentially regulated 210 genes, FunCat analysis indicated significant up-regulation of genes involved in fermentation, drug transport, pathogenesis and response to oxidative stress. Genes related to cytotoxicity were differentially regulated but the gliotoxin biosynthesis genes were down regulated over the time course, while Aspf1 was up-regulated at 9 h and 12 h. There was an up-regulation of genes in the subtelomeric regions of the genome as the interaction progressed. The genes up-regulated by iDC in the presence of A. fumigatus indicated that they were producing a pro-inflammatory response which was consistent with previous transcriptome studies of iDC interacting with A. fumigatus germ tubes. This study shows that A. fumigatus adapts to phagocytosis by iDCs by utilising genes that allow it to survive the interaction rather than just up-regulation of specific virulence genes.

Angiostatic and chemotactic activities of the CXC chemokine CXCL4L1 (platelet factor-4 variant) are mediated by CXCR3

AbstractWe investigated possible cellular receptors for the human CXC chemokine platelet factor-4 variant/CXCL4L1, a potent inhibitor of angiogenesis. We found that CXCL4L1 has lower affinity for heparin and chondroitin sulfate-E than platelet factor-4 (CXCL4) and showed that CXCL10 and CXCL4L1 could displace each other on microvascular endothelial cells. Labeled CXCL4L1 also bound to CXCR3A- and CXCR3B-transfectants and was displaced by CXCL4L1, CXCL4, and CXCL10. The CXCL4L1 anti-angiogenic activity was blocked by anti-CXCR3 antibodies (Abs) in the Matrigel and cornea micropocket assays. CXCL4L1 application in CXCR3−/− or in wild-type mice treated with neutralizing anti-CXCR3 Abs, resulted in reduced inhibitory activity of CXCL4L1 on tumor growth and vascularization of Lewis lung carcinoma. Furthermore, CXCL4L1 and CXCL4 chemoattracted activated T cells, human natural killer cells, and human immature dendritic cells (DCs). Migration of DCs toward CXCL4 and CXCL4L1 was desensitized by preincubation with CXCL10 and CXCL11, inhibited by pertussis toxin, and neutralized by anti-CXCR3 Abs. Chemotaxis of T cells, natural killer cells, and DCs is likely to contribute to the antitumoral action. However, the in vivo data indicate that the angiostatic property of CXCL4L1 is equally important in retarding tumor growth. Thus, both CXCR3A and CXCR3B are implicated in the chemotactic and vascular effects of CXCL4L1.

Neutrophil elastase treated dendritic cells promote the generation of CD4 +FOXP3 + regulatory T cells in vitro

We have previously shown that neutrophilic elastase converts human immature dendritic cells (DCs) into TGF-β secreting cells and reduces its allostimulatory ability. Since TGF-β has been involved in regulatory T cells (Tregs) induction we analyzed whether elastase or neutrophil-derived culture supernatant treated DCs induce CD4 +FOXP3 + Tregs in a mixed lymphocyte reaction (MLR). We found that elastase or neutrophil-derived culture supernatant treated DCs increased TGF-β and decreased IL-6 production. Together with this pattern of cytokines, we observed a higher number of CD4 +FOXP3 + cells in the MLR cultures induced by elastase or neutrophil-derived culture supernatant treated DCs but not with untreated DCs. The higher number of CD4 +FOXP3 + T cell population was not observed when the enzymatic activity of elastase was inhibited with an elastase specific inhibitor and also when a TGF-β1 blocking antibody was added during the MLR culture. The increased number of CD4 + that express FOXP3 was also seen when CD4 +CD25 - purified T cells were cocultured with the TGF-β producing DCs. Furthermore, these FOXP3 + T cells showed suppressive activity in vitro. These results identify a novel mechanism by which the tolerogenic DCs generated by elastase exposure contribute to the immune regulation and may be relevant in the pathogenesis of several lung diseases where the inflammatory infiltrate contains high numbers of neutrophils and high elastase concentrations.

LXR promotes the maximal egress of monocyte-derived cells from mouse aortic plaques during atherosclerosis regression

We have previously shown that mouse atherosclerosis regression involves monocyte-derived (CD68+) cell emigration from plaques and is dependent on the chemokine receptor CCR7. Concurrent with regression, mRNA levels of the gene encoding LXRalpha are increased in plaque CD68+ cells, suggestive of a functional relationship between LXR and CCR7. To extend these results, atherosclerotic Apoe-/- mice sufficient or deficient in CCR7 were treated with an LXR agonist, resulting in a CCR7-dependent decrease in plaque CD68+ cells. To test the requirement for LXR for CCR7-dependent regression, we transplanted aortic arches from atherosclerotic Apoe-/- mice, or from Apoe-/- mice with BM deficiency of LXRalpha or LXRbeta, into WT recipients. Plaques from both LXRalpha and LXRbeta-deficient Apoe-/- mice exhibited impaired regression. In addition, the CD68+ cells displayed reduced emigration and CCR7 expression. Using an immature DC line, we found that LXR agonist treatment increased Ccr7 mRNA levels. This increase was blunted when LXRalpha and LXRbeta levels were reduced by siRNAs. Moreover, LXR agonist treatment of primary human immature DCs resulted in functionally significant upregulation of CCR7. We conclude that LXR is required for maximal effects on plaque CD68+ cell expression of CCR7 and monocyte-derived cell egress during atherosclerosis regression in mice.

Jet exhaust particles alter human dendritic cell maturation

Among combustion-derived air pollutants, little is known about jet kerosene characteristics and effects. Particles yielded by experimental kerosene combustion in a jet engine were characterized with electron microscopy and X-ray energy dispersive spectroscopy. Immature human monocyte-derived dendritic cells were exposed for 18h to 10, 25 or 100μg/mL jet exhaust particles and/or Escherichia coli-derived endotoxin. Antigen-presenting and costimulation molecules (HLA DR, CD40, CD80, CD86, CD11c), tumor necrosis factor-α and interleukin-10 production were measured. The primary particles of jet exhaust are spherical (9.9nm), carbonaceous and exert an adjuvant effect on human monocyte-derived dendritic cell maturation in vitro. Concomitant particle and endotoxin stimulation induced a high cytokine production with low antigen-presenting molecules; particle contact prior to endotoxin contact led to an opposite phenotype. Finally, low cytokine production and high costimulation molecules were present when particle adjunction followed endotoxin contact. Jet exhaust particles act as adjuvants to endotoxin-induced dendritic cell maturation, suggesting possible implications for human health and a role for the time pattern of infectious and pollutant interplay.

Tracking Human Contact Allergens: From Mass Spectrometric Identification of Peptide-Bound Reactive Small Chemicals to Chemical-Specific Naive Human T-Cell Priming

Modification of proteins by reactive small chemicals is a key step in the activation of chemical-specific T cells in allergic contact dermatitis (ACD). However, an integrated approach to characterize both the precise nature of chemically modified proteins and the chemical-specific T cells is currently lacking. Here, we analyze the molecular conditions for adduct formation of the strong human contact sensitizer 2,4-dinitrochlorobenzene (DNCB) and its water-soluble form, 2,4-dinitrobenzenesulfonic acid (DNBS), with both an all amino acid-containing model peptide (± Cys) and the protein human serum albumin (HSA). Mass spectrometric detection and quantification revealed thiol-dependent peptide adduct formation at all pH values found in human skin layers. Highest modification rates were obtained with DNBS. Accordingly, DNBS- but not DNCB-modified human immature dendritic cells (iDC) induced in vitro primary human T-cell responses as did 2,4,6-trinitrobenzenesulfonic acid-modified iDC as measured by dinitrophenyl (DNP)- and trinitrophenyl (TNP)-specific T-cell proliferation and interferon gamma (IFN-γ) production in CD4(+) and CD8(+) T-cell subsets. Moreover, DNP-modified HSA protein effectively induced primary T-cell responses when processed by iDC. Thus, an integrated approach that combines efficient skin-related in chemico coupling analyses with an in vitro T-cell priming assay can be used to predict in vivo reactions of chemical contact allergens with extracellular and cellular proteins. This strategy supports the development of chemical-specific in vitro assays that are urgently required in predictive hazard identification and risk assessment of allergenic and nonallergenic chemicals.

Selective induction of host genes by MVA-B, a candidate vaccine against HIV/AIDS.

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.

Role of Glycogen Synthase Kinase 3 (GSK-3) in innate immune response of human immature dendritic cells toAspergillus fumigatus

Dysregulation of the Th1/Th2 cytokine balance and a switch to a Th2 immune response contribute to the development of and the unfavorable outcome from invasive aspergillosis (IA). We explore in this paper the role of glycogen synthase kinase 3 (GSK-3) in human immature dendritic cells (iDCs) relative to infection caused by A. fumigatus by the use of GSK-3 inhibitors (LiCl, SB415286) and RNA interference technology. In iDCs exposed to A. fumigatus germ tubes, inhibition of GSK-3 with LiCl or SB415286, as well as transfection with small interfering RNA, led to markedly elevated expression of the anti-inflammatory cytokine IL-10. In contrast, pro-inflammatory cytokine response was only partially regulated by GSK-3. Screening of patients after allogeneic stem cell transplantation (with or without IA) for the presence of genetic markers (rs334558, rs6438552) in the GSK-3 gene revealed no significant association with an increased risk for IA. In conclusion, GSK-3 might be involved in the regulation of the anti-inflammatory response of iDCs in the context of infections due to A. fumigatus, albeit the exact mechanisms have to be clarified in future experiments.

Frequent Calcium Oscillations Lead to NFAT Activation in Human Immature Dendritic Cells

Spontaneous Ca(2+) oscillations have been observed in a number of excitable and non-excitable cells, but in most cases their biological role remains elusive. In the present study we demonstrate that spontaneous Ca(2+) oscillations occur in immature human monocyte-derived dendritic cells but not in dendritic cells stimulated to undergo maturation with lipopolysaccharide or other toll like-receptor agonists. We investigated the mechanism and role of spontaneous Ca(2+) oscillations in immature dendritic cells and found that they are mediated by the inositol 1,4,5-trisphosphate receptor as they were blocked by pretreatment of cells with the inositol 1,4,5-trisphosphate receptor antagonist Xestospongin C and 2-aminoethoxydiphenylborate. A component of the Ca(2+) signal is also due to influx from the extracellular environment and may be involved in maintaining the level of the intracellular Ca(2+) stores. As to their biological role, our results indicate that they are intimately linked to the "immature" phenotype and are associated with the translocation of the transcription factor NFAT into the nucleus. In fact, once the Ca(2+) oscillations are blocked with 2-aminoethoxydiphenylborate or by treating the cells with lipopolysaccharide, NFAT remains cytoplasmic. The results presented in this report provide novel insights into the physiology of monocyte-derived dendritic cells and into the mechanisms involved in maintaining the cells in the immature stage.

Evidence that gC1qR and DC-SIGN associate on the surface of immature dendritic cells (136.24)

Abstract Complement protein C1q is involved in many immunoregulatory functions, such as modulating T-cell proliferation and regulating dendritic cell (DC) differentiation and maturation. C1q has at least two multi-functional cell surface receptors, cC1qR and gC1qR. While these receptors ostensibly lack a direct conduit into intracellular elements, they are known to form docking/signaling complexes with transmembrane partners. DC-SIGN (dendritic cell-specific-ICAM-grabbing-non integrin) is a C-type lectin expressed on the surface of DCs and involved in T-cell activation and endocytosis. Because SIGN-R1, a C-type lectin related to DC-SIGN has been shown to bind directly to C1q, we investigated whether DC-SIGN also binds to C1q and if DC-SIGN may act as a transmembrane partner for gC1qR and/or cC1qR. Using flow cytometry, we found that DC-SIGN, gC1qR and cC1qR are all present on the surface of human immature DCs (iDC) grown in culture. While antigen-capture ELISA experiments using iDC lysates showed that C1q, gC1qR and cC1qR directly or indirectly associate with DC-SIGN, immunofluorescence microscopy revealed that C1q and gC1qR, but not cC1qR co-localize with DC-SIGN on the surface of iDCs. The data suggest that DC-SIGN and gC1qR may directly associate on the surface of iDCs and regulate DC differentiation in a stage specific manner. Since both gC1qR and DC-SIGN have been shown to bind HIV, it is possible that the two receptors may play a role in the pathogenesis of HIV-AIDS.

Effects of glycation of the model food allergen ovalbumin on antigen uptake and presentation by human dendritic cells

Advanced glycation endproducts (AGEs) of food proteins resulting from the Maillard reaction after cooking or heating may have particular importance in food allergy. The underlying immunological mechanisms are only poorly understood. The aim of the study was to examine the effects of AGE derived from the model food allergen ovalbumin (AGE-OVA) on dendritic cells (DCs), their immunostimulatory capacity and the T-cell response compared with regular OVA. For this purpose, human immature DCs were exposed to fluorescein isothiocyanate (FITC)-labelled AGE-OVA and FITC-labelled regular OVA and uptake was analysed by flow cytometry and fluorescence microscopy. Furthermore, autologous CD4(+) T-cell proliferation and cytokine production induced by mature DCs loaded with AGE-OVA were compared with those induced by mature DCs loaded with OVA. Finally, expression of the receptor for advanced glycation endproducts (RAGE) and activation of the transcription factor nuclear factor (NF)-kappaB by AGE were investigated. Internalization of FITC-AGE-OVA by immature DCs was significantly increased compared with FITC-OVA. Blocking the mannose receptor, macropinocytosis or the scavenger receptor strongly reduced uptake of both FITC-OVA and FITC-AGE-OVA. In a comparison of CD4(+) T cells co-cultured with AGE-OVA-loaded mature DCs versus those co-cultured with OVA-loaded mature DCs, AGE-OVA DCs were found to produce more interleukin (IL)-6 and to induce a stronger T helper type 2 (Th2) and a weaker Th1 cytokine response, while there was no difference in proliferation of CD4(+) T cells. The expression of RAGE was higher on immature DCs compared with mature DCs. AGE-OVA-exposed immature DCs showed a stronger expression of RAGE and activation of the transcription factor NF-kappaB compared with OVA-loaded immature DCs. Our data indicate that AGE-OVA may be more immunogenic/allergenic than regular OVA.

Antibody-assisted enhancement of biological activities of CXCL14 in human monocytic leukemia-derived THP-1 cells and high fat diet-induced obese mice

CXCL14 is a CXC-type chemokine acting on tissue macrophages, immature dendritic cells, natural killer cells, and epithelial tumor cells. It also serves as a metabolic regulator in obese mice by blunting insulin activity. In contrast to other CXC chemokines, it remains to be clarified how CXCL14 activates its putative receptors on the cell surface and whether it induces chemokinesis. This is mainly due to the insufficient sensitivity of currently available bioassays for CXCL14. In this study, we found that the anti-CXCL14 monoclonal antibody, MAB730, remarkably enhances the activities of CXCL14 in human monocytic leukemia-derived THP-1 cells and immature dendritic cells. MAB730 augmented CXCL14-mediated chemotaxis and chemokinesis with distinct dose requirement. Chemotaxis inducing activity was retained in the MAB730 F(ab')2 fraction, but not in the Fab fraction, implying that ligand dimerization is involved in the MAB730-assisted enhancement of CXCL14 activity. In addition, MAB730 was more efficient than heparin at inhibiting CXCL14 binding to low affinity receptors on THP-1 cells. Finally, in vivo administration of MAB730 antibody into high fat diet-induced obese mice increased whole body insulin resistance and glucose intolerance. These unique properties of MAB730 will be useful for elucidating the molecular mechanism of cellular responses elicited by CXCL14.

Frequent Calcium Oscillations Lead to NFAT Activation in Human Immature Dendritic Cells

Spontaneous Ca2+ oscillations have been reported in certain types of excitable and non-excitable cells. However, the precise molecular mechanism underlying these events and their biological role(s) remain unclear. In the present study we demonstrate for the first time that spontaneous Ca2+ oscillations occur in immature human monocyte-derived dendritic cells and that upon receiving maturation signals via TLRs, the cells lose the high frequency Ca2+ oscillations. We investigated the mechanism and role of the Ca2+ oscillations in immature dendritic cells and found that the inositol-1,4,5-trisphosphate receptor is essential, since oscillations were blocked by pre-treatment of cells with the inositol-1,4,5-trisphosphate receptor antagonist Xestospongin C and 2-APB. A component of the Ca2+ signal is also due to influx from the extracellular environment and may be involved in refilling the intracellular Ca2+ stores. As to their biological role, our results indicate that they are intimately linked to the “immature” phenotype and are associated with the translocation of the transcription factor NFAT into the nucleus. In fact, blocking the Ca2+ oscillations with 2-APB or treating the cells with LPS, leading then to undergo maturation, caused NFAT to remain in the cytoplasm. The results presented in this report provide novel insights into the physiology of immature dendritic cells and into the signaling process(es) controlling their maturation.

Mouse ChemR23 Is Expressed in Dendritic Cell Subsets and Macrophages, and Mediates an Anti-Inflammatory Activity of Chemerin in a Lung Disease Model

Chemerin is the ligand of the ChemR23 receptor and a chemoattractant factor for human immature dendritic cells (DCs), macrophages, and NK cells. In this study, we characterized the mouse chemerin/ChemR23 system in terms of pharmacology, structure-function, distribution, and in vivo biological properties. Mouse chemerin is synthesized as an inactive precursor (prochemerin) requiring, as in human, the precise processing of its C terminus for generating an agonist of ChemR23. Mouse ChemR23 is highly expressed in immature plasmacytoid DCs and at lower levels in myeloid DCs, macrophages, and NK cells. Mouse prochemerin is expressed in most epithelial cells acting as barriers for pathogens but not in leukocytes. Chemerin promotes calcium mobilization and chemotaxis on DCs and macrophages and these functional responses were abrogated in ChemR23 knockout mice. In a mouse model of acute lung inflammation induced by LPS, chemerin displayed potent anti-inflammatory properties, reducing neutrophil infiltration and inflammatory cytokine release in a ChemR23-dependent manner. ChemR23 knockout mice were unresponsive to chemerin and displayed an increased neutrophil infiltrate following LPS challenge. Altogether, the mouse chemerin/ChemR23 system is structurally and functionally conserved between human and mouse, and mouse can therefore be considered as a good model for studying the anti-inflammatory role of this system in the regulation of immune responses and inflammatory diseases.

Enterovirus 71 Infection of Human Dendritic Cells

Enterovirus 71 (EV71) causes death and long-term neurologic sequelae in hundreds of thousands of young children, but its pathogenesis remains elusive. Dendritic cells (DCs) play a crucial role in antiviral immunity by functioning as professional antigen-presenting cells to prime T cells and by secreting cytokines to modulate immune responses. Here, we show that EV71 productively infected human immature DCs and expressed viral antigen in DCs. EV71 entry into DCs was partially mediated by DC-SIGN. Further analyses revealed that EV71 increased the viability, activation, release of cytokines, interleukin-6, interleukin-12, and tumor necrosis factor-alpha in DCs. Moreover, EV71 enabled DCs to stimulate T-cell proliferation. Collectively, these findings suggest that EV71 infection of human DCs in vivo is very likely to elicit protective immunity, because in infected mice, both T cells and IL-6 function to reduce mortality.