Differential processing of autoantigens in lysosomes from human monocyte-derived and peripheral blood dendritic cells

Dendritic cells (DC) initiate immunity and maintain tolerance. Although in vitro-generated DC, usually derived from peripheral blood monocytes (MO-DC), serve as prototype DC to analyze the biology and biochemistry of DC, phenotypically distinct primary types of DC, including CD1c-DC, are present in peripheral blood (PB-DC). The composition of lysosomal proteases in PB-DC and the way their MHC class II-associated Ag-processing machinery handles a clinically relevant Ag are unknown. We show that CD1c-DC lack significant amounts of active cathepsins (Cat) S, L, and B as well as the asparagine-specific endopeptidase, the major enzymes believed to mediate MHC class II-associated Ag processing. However, at a functional level, lysosomal extracts from CD1c-DC processed the multiple sclerosis-associated autoantigens myelin basic protein and myelin oligodendrocyte glycoprotein in vitro more effectively than MO-DC. Although processing was dominated by CatS, CatD, and asparagine-specific endopeptidase in MO-DC, it was dominated by CatG in CD1c-DC. Thus, human MO-DC and PB-DC significantly differ with respect to their repertoire of active endocytic proteases, so that both proteolytic machineries process a given autoantigen via different proteolytic pathways.     

Gene expression analysis in human monocytes, monocyte-derived dendritic cells, and alpha-galactosylceramide-pulsed monocyte-derived dendritic cells.

In vitro proliferation and functional activation of V alpha 24NKT cells following stimulation with alpha-galactosylceramide (alpha-GalCer)-pulsed dendritic cells (DCs) have been observed. Because little is known about the molecular events on DCs following interaction with alpha-GalCer, we performed gene expression profiling of 2400 genes in monocytes and monocyte-derived immature DCs pulsed with alpha-GalCer (alpha-GalCer-imDCs). Overall, the expression levels of 48 genes were up-regulated and 28 were down-regulated in alpha-GalCer-imDCs. Semiquantitative RT-PCR analysis on monocytes, imDCs, alpha-GalCer-imDCs, and mature DCs confirmed the up- and down-regulation of the mRNA expression levels of 28 selected genes. Notably, we identified the specific up-regulation of mRNA expression levels of ribonuclease A and collapsin response mediator protein upon the stimulation of imDC with alpha-GalCer, suggesting a novel immunomodulating effect of alpha-GalCer on imDCs. In this study, we used imDCs prepared by culturing of monocytes with GM-CSF and IL-4 for 5 days and mDCs prepared by further culturing of imDCs with TNF alpha for two extra days.     

Morphine reciprocally regulates IL-10 and IL-12 production by monocyte-derived human dendritic cells and enhances T cell activation

We evaluated the effect of morphine on human dendritic cells (DCs). Interestingly, immature DCs were found to express all 3 (mu, kappa, delta) opioid receptors on the cell surface. Chronic morphine treatment (10(-8) to 10(-12) M) during the development of DCs from monocytes augmented LPS-induced upregulation of HLA-DR, CD86, CD80, and CD83 and increased the T cell stimulatory capacity of DCs, which could be inhibited by naloxone, an opioid receptor antagonist. The change in surface phenotype was paralleled by a p38 MAPK-dependent decrease in IL-10 and increase in IL-12 secretion. Our data indicate that morphine exerts an immunostimulatory effect by modulating LPS-induced DC maturation.     

TGF-beta 1 reciprocally controls chemotaxis of human peripheral blood monocyte-derived dendritic cells via chemokine receptors

We examined the effect of TGF-beta 1 on the chemotactic migratory ability of human monocyte-derived dendritic cells (DCs). Treatment of immature DCs with TGF-beta 1 resulted in increased expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXC chemokine receptor-4 (CXCR-4), which were concomitant with enhanced chemotactic migratory responses to their ligands, RANTES (for CCR-1, CCR-3, and CCR-5), macrophage-inflammatory protein-3 alpha (MIP-3 alpha) (for CCR-6), or stromal cell-derived growth factor-1 alpha (for CXCR-4). Ligation by TNF-alpha resulted in down-modulation of cell surface expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and the chemotaxis for RANTES, MIP-3 alpha, and stromal cell-derived growth factor-1 alpha, whereas this stimulation up-regulated the expression of CCR-7 and the chemotactic ability for MIP-3beta. Stimulation of mature DCs with TGF-beta 1 also enhanced TNF-alpha-induced down-regulation of the expressions of CCR-1, CCR-3, CCR-5, CCR-6, and CXCR-4, and chemotaxis to their respective ligands, while this stimulation suppressed TNF-alpha-induced expression of CCR-7 and chemotactic migratory ability to MIP-3 beta. Our findings suggest that TGF-beta 1 reversibly regulates chemotaxis of DCs via regulation of chemokine receptor expression.     

Autocrine activation-induced cell death of T cells by human peripheral blood monocyte-derived CD4+ dendritic cells

Mature T cells activated by antigen (Ag)-presenting cells are subject to various downmodulatory processes designed to maintain T cell homeostasis. Here we describe experiments in which mature T cells were subjected to apoptosis following stimulation with CD4(+) dendritic cells (DCs) during Ag presentation. The proliferative response of allogeneic T cells was increased by DCs at stimulator to responder (S/R) ratios ranging from 10(-3) to 1, whereas this response was decreased at S/R ratios ranging from 2 to 10. Allogeneic T cells stimulated with DCs at an S/R ratio of 5 underwent apoptosis, whereas this event was not observed in allogeneic T cells stimulated with DCs at an S/R ratio of 0.5. Stimulation of T cells with DCs at an S/R ratio of 5 induced a higher level of expression of CD95 ligand (CD95L) than stimulation of T cells cultured with DCs at an S/R ratio of 0.5, whereas similar levels of expression of CD28 and CD154 were observed in both cells. The abortive proliferation of mature T cells stimulated with DCs was prevented by blocking the CD95-CD95L system. Our results suggest that the CD4(+) DCs play counterregulatory roles in dictating T cell responses during Ag presentation.     

Enhancement of migratory and aggregate activities of human peripheral blood monocyte-derived dendritic cells by stimulation with RANTES

We examined the effects of various chemokines on the functional activation of granulocyte-macrophage colony-stimulating factor (GM-CSF) plus interleukin-4 (IL-4)-generated human peripheral blood monocyte-derived immature dendritic cells (iDC). Stimulation of iDC with regulated on activation normal T cell expressed and secreted (RANTES) resulted in the promotion of their chemotactic migratory capacity in response to RANTES when compared with that of unstimulated cells. TNF-alpha induced a homotypic aggregated cluster formation of iDC in a dose-dependent manner, whereas the combination of TNF-alpha and RANTES exhibited more potent induction. IDC stimulated with RANTES were more efficient than unstimulated iDC in the production of endogenous RANTES. Treatment of iDC with the combination of TNF-alpha and RANTES was just little effective for the enhancement of allogeneic T-cell stimulatory capacity as compared with that of TNF-alpha treated iDC. These results suggest that endogenous secretions of RANTES from iDC stimulated with RANTES be potentially involved in RANTES-induced changes of properties with respect to morphology and function.     

IL-10 inhibits cysteinyl leukotriene-induced activation of human monocytes and monocyte-derived dendritic cells

The immunoregulatory cytokine IL-10 plays an essential role in down-modulating adaptive and innate immune responses leading to chronic inflammatory diseases. In contrast, cysteinyl leukotrienes (cysLTs), important proinflammatory mediators of cell trafficking and innate immune responses, are thought to enhance immune reactions in the pathogenesis of diseases, such as bronchial asthma, atherosclerosis, and pulmonary fibrosis. The aim of this study was to determine the IL-10 regulatory role in cysLT-induced activation of human monocytes and monocyte-derived dendritic cells. Herein we show that cysLT-induced activation and chemotaxis of human monocytes and monocyte-derived immature dendritic cells (iDC) are inhibited by IL-10 pretreatment. IL-10 down-regulated cysLT type 1 and 2 receptors' mRNA in a time- and concentration-dependent fashion. cysLT-induced activation of monocytes and iDCs measured by intracellular calcium flux and immediate-early gene expression (FBJ murine osteosarcoma viral oncogen homolog B and early growth response-2) was potently decreased by IL-10 and by the cysLT antagonist MK571. Chemotaxis of monocytes and iDCs to increasing concentrations of leukotriene D(4) (LTD(4)) was also inhibited by IL-10. LTD(4) enhanced iDC migration in response to CCL5. IL-10 selectively inhibited LTD(4)-induced chemotaxis without affecting migration to CCL5. These data indicate that cysLT-induced activation of human monocytes and dendritic cells may be specifically inhibited by IL-10, suggesting a direct link between the 5-lipoxygenase proinflammatory pathway and IL-10 regulatory mechanisms. Antileukotriene therapies may reproduce some regulatory mechanisms played by IL-10 in inflammatory processes.     

Expression of high-affinity IgE receptor on human peripheral blood dendritic cells in children

Background

In a mouse model of viral induced atopic disease, expression of FcεRI on dendritic cells is critical. While adult human conventional (cDC) and plasmacytoid (pDC) dendritic cells have been shown to express FcεRI, it is not known if this receptor is expressed in childhood and how its expression is governed by IgE.

Methods

Following informed consent of subjects (n = 27, aged 12–188 months), peripheral blood was stained for surface expression of CD19, ILT7, CD1c, IgE, FcεRI and analyzed by flow cytometry (cDC: CD19⁻ ILT7⁻ CD1c⁺; pDC: CD19⁻ ILT7⁺ CD1c⁻). Total and specific serum IgE levels to food and inhalant allergens were determined by ImmunoCAP, and the relationship between FcεRI expression on dendritic cells and sensitization, free IgE, cell bound IgE, and age was determined.

Results

Independent of sensitization status, FcεRI expression was noted on cDC and pDC as early as 12 months of age. Serum IgE level correlated with expression of FcεRI on cDC, but not pDC. Based on the concentration of IgE, a complex relationship was found between surface bound IgE and expression of FcεRI on cDC. pDC exhibited a linear relationship of FcεRI expression and bound IgE that was consistent through all IgE concentrations.

Conclusions

In children, FcεRI expression on cDC and pDC is modulated differently by serum and cell bound IgE. IgE governance of FcεRI expression on cDC depends upon a complex relationship. Further studies are needed to determine the functional roles of FcεRI on cDC and pDC.     

IFN-alpha and IL-12 induce IL-18 receptor gene expression in human NK and T cells

IL-18 is a proinflammatory cytokine that enhances innate and specific Th1 immune responses. During microbial infections, IL-18 is produced by activated macrophages. IL-18 exerts its effects in synergy with IFN-alpha or IL-12 to induce IFN-gamma. Here we show that in human NK and T cells IFN-alpha and IL-12 strongly up-regulate mRNA expression of the IL-18R components, accessory protein-like (AcPL) and IL-1R-related protein (IL-1Rrp). In addition, IFN-alpha enhanced the expression of MyD88, an adaptor molecule involved in IL-18 signaling. Pretreatment of T cells with IFN-alpha or IL-12 enhanced IL-18-induced NF-kappaB activation and sensitized the cells to respond to lower concentrations of IL-18. AcPL and IL-1Rrp genes were strongly expressed in T cells polarized with IL-12, whereas in IL-4-polarized cells these genes were expressed at very low levels, indicating that AcPL and IL-1Rrp genes are preferentially expressed in Th1 cells. In conclusion, the results suggest that IFN-alpha and IL-12 enhance innate as well as Th1 immune response by inducing IL-18R expression.     

Analysis of proteomic profiles and functional properties of human peripheral blood myeloid dendritic cells,monocyte-derived dendritic cells and the dendritic cell-like KG-1 cells reveals distinct characteristics

Background  

Dendritic cells (DCs) are specialized antigen presenting cells that play a pivotal role in bridging innate and adaptive immune responses. Given the scarcity of peripheral blood myeloid dendritic cells (mDCs) investigators have used different model systems for studying DC biology. Monocyte-derived dendritic cells (moDCs) and KG-1 cells are routinely used as mDC models, but a thorough comparison of these cells has not yet been carried out, particularly in relation to their proteomes. We therefore sought to run a comparative study of the proteomes and functional properties of these cells.     

Human monocyte-derived dendritic cells turn into foamy dendritic cells with IL-17A

Interleukin 17A (IL-17A) is a proinflammatory cytokine involved in the pathogenesis of chronic inflammatory diseases. In the field of immunometabolism, we have studied the impact of IL-17A on the lipid metabolism of human in vitro-generated monocyte-derived dendritic cells (DCs). Microarrays and lipidomic analysis revealed an intense remodeling of lipid metabolism induced by IL-17A in DCs. IL-17A increased 2–12 times the amounts of phospholipids, cholesterol, triglycerides, and cholesteryl esters in DCs. Palmitic (16:0), stearic (18:0), and oleic (18:ln-9c) acid were the main fatty acid chains present in DCs. They were strongly increased in response to IL-17A while their relative proportion remained unchanged. Capture of extracellular lipids was the major mechanism of lipid droplet accumulation, visualized by electron microscopy and Oil Red O staining. Besides this foamy phenotype, IL-17A induced a mixed macrophage-DC phenotype and expression of the nuclear receptor NR1H3/liver X receptor-α, previously identified in the context of atherosclerosis as the master regulator of cholesterol homeostasis in macrophages. These IL-17A-treated DCs were as competent as untreated DCs to stimulate allogeneic naive T-cell proliferation. Following this first characterization of lipid-rich DCs, we propose to call these IL-17A-dependent cells “foamy DCs” and discuss the possible existence of foamy DCs in atherosclerosis, a metabolic and inflammatory disorder involving IL-17A.     

Standardized generation of fully mature p70 IL-12 secreting monocyte-derived dendritic cells for clinical use

Dendritic cells (DC) have been shown to be efficient antigen-presenting cells (APC) and, as such, could be considered ideal candidates for cancer immunotherapy. Immature DC (iDC) efficiently capture surrounding antigens; however, only mature DC (mDC) prime naive T lymphocytes. Clinical trials using DC-based tumor vaccines have achieved encouraging, but limited, success, possibly due to the use of immature or incompletely mature DC. Thus, it was apparent that a method capable of generating large numbers of fully functional iDC, their pulsing with desired form of tumor antigens and the subsequent complete and reproducible maturation of iDC is needed. Therefore, we compared two different methods of producing large numbers of iDC. Both protocols yielded comparable numbers of cells with an iDC phenotype with phagocytic function. We next determined which of the clinically applicable activators could induce the complete and reproducible maturation of DC, in order to define the most suitable combination for future clinical trials. Only a combination of TNFalpha + Poly (I:C), or a previously described cytokine cocktail of TNFalpha + IL-1beta + IL-6 + prostaglandin E2, induced the complete activation of the whole DC population, as assessed by the cell surface expression of CD83 and costimulatory molecules. The matured DC were functionally superior to iDC in their ability to stimulate the proliferation of allogeneic lymphocytes and autologous keyhole limpet hemocyanin (KLH)-specific T lymphocytes. Furthermore, only the combination of TNFalpha + Poly (L:C) activated DC to produce large amounts of biologically active p70 IL-12. Thus DC maturation by TNFalpha + Poly (I:C) could efficiently bias T cell response towards Th1 response. Implementation of our results into clinical protocols used for DC generation could be beneficial for future immunotherapy trials.     

IL-15 activated human peripheral blood dendritic cell kill allogeneic and xenogeneic endothelial cells via apoptosis

IL-15 is a pleotropic cytokine, which plays an important role in natural killer (NK) cell activity, T cell proliferation, and T cell cytotoxic activity. Dendritic cells (DCs) are the major antigen presenting cells in the immune system and presumed to play an important role in immune recognition of allo and xenotransplantation. We showed that IL-15 activated human peripheral blood DC is cytotoxic to human and porcine aortic endothelial cells. Unlike DCs, CD14+ monocytes show no cytotoxicity against the endothelial cells. This cytotoxic potential of IL-15 activated DC against endothelial cells is dose dependent and increases significantly upon treatment of endothelial cells with inflammatory cytokines like TNF-α or IFN-γ. The cytotoxic potential of IL-15 activated DC is associated with apoptosis of endothelial cells, as indicated by the increased Annexin V staining, caspase activation and loss of mitochondrial membrane potential. Further it was observed that DC mediated cytotoxicity against endothelial cell is mediated via granzyme B possibly secreted by the activated DCs.     

Autoregulation of human monocyte-derived dendritic cell maturation and IL-12 production by cyclooxygenase-2-mediated prostanoid production

PG added to cell culture profoundly affect the in vitro maturation and function of monocyte-derived dendritic cells (MDC). Because unstimulated monocytes express cyclooxygenase (COX)-1, and COX-2 when activated, we examined whether MDC express these enzymes and produce prostanoids that autoregulate maturation and IL-12 production. Immature MDC (I-MDC) and mature MDC express COX-1, but, unlike monocytes, both MDC populations constitutively express COX-2. However, COX-2 regulation in both MDC populations differs from monocytes, as IL-4 does not suppress enzyme expression. COX-2 is functional in MDC as a specific inhibitor, NS-398, significantly reduces PGE(2) production. I-MDC undergoing maturation with soluble CD40 ligand (sCD40L) increase PGE(2) synthesis, but prostanoid synthesis is switched to COX-1. However, with IFN-gamma present, sCD40L-stimulated PG metabolism is redirected to COX-2, and PGE(2) synthesis increases severalfold. Endogenous PG production by MDC does not regulate CD40, CD80, CD86, or HLA DR expression; however, it does promote MDC maturation, as NS-398 significantly reduces CD83 expression in I-MDC matured with sCD40L/IFN-gamma. PG produced through COX-2 also autoregulate IL-12, but the effects are dependent on the MDC maturation state. Blocking COX-2 reduces I-MDC secretion of IL-12p40, whereas it increases IL-12p40 and p70 production by maturing MDC. COX-2-mediated PG production impacts MDC function as maturing these cells in the presence of NS-398 yields MDC that stimulate significantly more IFN-gamma in an allogeneic mixed lymphocyte response than MDC matured without this inhibitor. These studies demonstrate that MDC express both COX isoforms constitutively and produce prostanoids, which autoregulate their maturation and function.     

Bidirectional negative regulation of human T and dendritic cells by CD47 and its cognate receptor signal-regulator protein-alpha: down-regulation of IL-12 responsiveness and inhibition of dendritic cell activation

Proinflammatory molecules, including IFN-gamma and IL-12, play a crucial role in the elimination of causative agents. To allow healing, potent anti-inflammatory processes are required to down-regulate the inflammatory response. In this study, we first show that CD47/integrin-associated protein, a ubiquitous multispan transmembrane protein highly expressed on T cells, interacts with signal-regulator protein (SIRP)-alpha, an immunoreceptor tyrosine-based inhibition motif-containing molecule selectively expressed on myelomonocytic cells, and next demonstrate that this pair of molecules negatively regulates human T and dendritic cell (DC) function. CD47 ligation by CD47 mAb or L-SIRP-alpha transfectants inhibits IL-12R expression and down-regulates IL-12 responsiveness of activated CD4(+) and CD8(+) adult T cells without affecting their response to IL-2. Human CD47-Fc fusion protein binds SIRP-alpha expressed on immature DC and mature DC. SIRP-alpha engagement by CD47-Fc prevents the phenotypic and functional maturation of immature DC and still inhibits cytokine production by mature DC. Finally, in allogeneic MLR between mDC and naive T cells, CD47-Fc decreases IFN-gamma production after priming and impairs the development of a Th1 response. Therefore, CD47 on T cells and its cognate receptor SIRP-alpha on DC define a novel regulatory pathway that may be involved in the maintenance of homeostasis by preventing the escalation of the inflammatory immune response.     

Activation of toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10

Mammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1alpha+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity.     

Decreased response to IL-12 and IL-18 of peripheral blood cells in rheumatoid arthritis

Inflamed synovium of rheumatoid arthritis (RA) has been associated with a T helper (Th)1 cytokine profile but the blood situation remains to be clarified. We studied the differential IFN-γ producing activity of peripheral blood mononuclear cells (PBMCs) from RA patients (RA-PBMCs) and from healthy controls (H-PBMCs) in response to IL-12 and IL-18. RA-PBMCs had a decreased IFN-γ production in response to IL-12 and IL-18 when compared with H-PBMCs. RA-PBMCs activated with phytohemagglutinin and phorbol 12-myristate 13-acetate showed an increased sensitivity to IL-12 and IL-18, but still the RA-PBMC response was lower. IL-18 increased IL-12-stimulated IFN-γ production from RA synovium cells obtained after collagenase digestion more effectively than that of RA- or H-PBMCs. A specific inhibitor of IL-18 bioactivity, IL-18-binding protein (IL-18BP), down-regulated IL-12-induced IFN-γ production by RA- or H-PBMCs and had a remarkable effect on RA synovium cells. In conclusion, RA disease combines a polarized immune response with an active Th1 in inflamed joints and a reduced Th1 pattern in peripheral circulation.