Late dendritic cells are still able to evoke a potent alloreactive CTL response

On exposure to maturation stimuli, immature dendritic cells (DCs) undergo changes that turn them into potent amplifiers of innate immunity and into antigen-presenting cells (APCs) able to prime na?ve T cells. However, their progression through the maturation process is very rapid and finally ends in apoptosis. The aim of our study was to investigate the importance of the maturation stage of DCs, defined by morphology, expression of surface markers and IL-12 production, for their immunostimulatory capacity. DCs were matured with LPS, monocyte-conditioned medium (MCM) or TNF-alpha, sampled several times during a 3-day long maturation period and used as stimulators of allogeneic T cells over a wide range of DC/T cell ratios. T-cell response was assessed by cell proliferation, CTL generation and IFN-gamma production. Our results indicate that the in vitro T cell response is determined mainly by the level of expression of co-stimulatory molecules on DCs and the DC/T cell ratio in the culture. Thus, DCs matured for over 20h, with high expression of co-stimulatory molecules, can still induce a potent CTL response at DC/T cell ratios of 1:10 and 1:20, although their IL-12 production, as well as their ability to induce IFN-gamma production by T cells, are both decreased. In contrast, the CTL response at DC/T cell ratios of 1:2 and 1:5 can be profoundly decreased. Notably, the proportion of proliferating CD4+ T cells in these cultures is reduced. This could well be the reason for the absence of CTL response, since we showed that, even in the case of high expression of co-stimulatory molecules on DCs, generation of CTLs still depends on CD4+ T cells. Our study emphasizes the importance of strong expression of co-stimulatory molecules on DCs and of their ability to activate CD8+ and CD4+ T cells concomitantly in order to initiate a potent cell-mediated immune response. We therefore suggest that a combination of early DCs, which are strong producers of cytokines, and late DCs, which have high expression of co-stimulatory molecules, could prove beneficial in the attempt to initiate in vitro and in vivo cell-mediated immune responses for therapeutic purposes.     

Endotoxin can induce MyD88-deficient dendritic cells to support T(h)2 cell differentiation

Toll-like receptor (TLR) signaling activates dendritic cells (DC) to secrete proinflammatory cytokines and up-regulate co-stimulatory molecule expression, thereby linking innate and adaptive immunity. A TLR-associated adapter protein, MyD88, is essential for cytokine production induced by TLR. However, in response to a TLR4 ligand, lipopolysaccharide (LPS), MyD88-deficient (MyD88(-/-)) DC can up-regulate co-stimulatory molecule expression and enhance their T cell stimulatory activity, indicating that the MyD88-independent pathway through TLR4 can induce some features of DC maturation. In this study, we have further characterized function of LPS-stimulated, MyD88(-/-) DC. In response to LPS, wild-type DC could enhance their ability to induce IFN-gamma production in allogeneic mixed lymphocyte reaction (alloMLR). In contrast, in response to LPS, MyD88(-/-) DC augmented their ability to induce IL-4 instead of IFN-gamma in alloMLR. Impaired production of T(h)1-inducing cytokines in MyD88(-/-) DC cannot fully account for their increased T(h)2 cell-supporting ability, because absence of T(h)1-inducing cytokines in DC caused impairment of IFN-gamma, but did not lead to augmentation of IL-4 production in alloMLR. In vivo experiments with adjuvants also revealed T(h)2-skewed immune responses in MyD88(-/-) mice. These results demonstrate that the MyD88-independent pathway through TLR4 can confer on DC the ability to support T(h)2 immune responses.     

Regulatory role of nitric oxide on monocyte-derived dendritic cell functions.

Nitric oxide (NO) has an established role in the defense against bacterial infections and exerts multiple modulatory activities on both inflammatory and immune responses. However, the relevance of NO on dendritic cell (DC) functions has been poorly investigated. In this study, we found that addition of the NO donor S-nitrosoglutathione (GSNO) to monocyte-derived DCs matured by lipopolysaccharide (LPS) or soluble CD40 ligand led to a decreased capacity to activate naive allogeneic T cells but a more prominent Th1 polarization, with increased interferon-gamma (IFN-gamma) secretion and reduced interleukin-5 (IL-5) release. The presence of GSNO during maturation of DCs caused a reduced expression of surface CD86, whereas CD80, CD83, and MHC molecule expression was not affected. Moreover, GSNO induced a dose-dependent decrease of IL-10 and enhancement of tumor necrosis factor-alpha (TNF-alpha) release from mature DCs. In parallel, a marked reduced production of IL-12 p40 subunit but no significant perturbation of the bioactive IL-12 p70 production was observed. Finally, GSNO significantly reduced the release of IP-10/CXCL10 and RANTES/CCL5 but not IL-8/CXCL8 by mature DCs. Although GSNO can strengthen the capacity of mature DCs to induce type 1 polarization of T lymphocytes, our data suggest that it elicits distinct anti-inflammatory functions, eventually reducing T lymphocyte proliferation and recruitment.     

Mature dendritic cells differentiated in the presence of interferon-beta and interleukin-3 prime functional antigen-specific CD8 T cells

Dendritic cell (DC)-based immunization represents a promising approach for the immunotherapy of cancer. The optimal conditions required to prepare DCs remain to be defined. Monocytes incubated in the presence of interferon (IFN)-beta and interleukin (IL)-3 give rise to a distinct type of DCs (IFN-beta/IL-3 DCs) that are particularly efficient at eliciting IFN-gamma and IL-5 production by allogeneic helper T cells. We assessed the capacity of this new type of DCs to prime antigen-specific naive CD8(+) T cells and compared them to the conventional DCs differentiated in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4 (GM-CSF/IL-4 DCs). We demonstrate that IFN-beta/IL-3 DCs matured by TLR3 or CD40 ligation efficiently prime Melan-A(26-35)-specific CD8(+) T cells in vitro, at a similar level as GM-CSF/IL-4 DCs. Activated antigen-specific CD8(+) T cells produced IFN-gamma and displayed potent cytotoxic activity against peptide-pulsed target cells. Expansion of CD8(+) T cell numbers was generally higher following priming with CD40-L than with polyinosinic-polycytidylic acid (poly I:C) matured DCs. Cytolytic activity was induced by both maturing agents. These data indicate that IFN-beta/IL-3 DCs represent a promising cell population for the immunotherapy of cancer.     

CD44 mobilization in allogeneic dendritic cell-T cell immunological synapse plays a key role in T cell activation

CD44 is involved in several biological processes owing to its dual role as a cell adhesion and signaling molecule. In an allogeneic dendritic cell (DC)-T cell interaction model, we show here that CD44 gets clustered at the contact between T cells with mature but not immature DCs. Also, CD44 colocalized with lipid rafts at the immunological synapse (IS). Using DCs or T cells derived from CD44-deficient mice, we observed that the presence of CD44 on DCs and T cells is important for the formation of DC-T cell tight conjugates. However, deficiency of CD44 on DCs but not T cells affected the functional IS, as indicated by decreased phosphotyrosine and protein kinase C-theta enrichment at the synapse. Also, CD44-deficient DCs induced significantly decreased proliferation as well as IL-2 and IFN-gamma production from allogeneic T cells. The polarization of CD44 at the synapse was also noted in an antigen (OVA)-specific, syngeneic DC-T cell interaction using OVA-specific T cells derived from OT-II mice. It was believed that large molecules such as CD44 were excluded from the IS. Results presented here show for the first time that CD44 is recruited to the IS during allogeneic DC and T cell interactions and plays an important role in subsequent T cell activation.     

Interleukin-4 selectively inhibits interleukin-2 secretion by lipopolysaccharide-activated dendritic cells

Dendritic cells (DCs) generated in vitro from bone marrow precursors using granulocyte-macrophage colony-stimulating factor (GM-CSF) secrete interleukin-2 (IL-2) upon activation, an event probably associated to the initiation of adaptive immune responses. Additionally, they produce IL-12, a cytokine related to T-cell polarization. To analyse the effect of IL-4 on DC differentiation and function, we assessed the capacity of murine bone marrow dendritic cells (BMDCs) differentiated with GM-CSF in the presence or absence of IL-4 to produce IL-2 and IL-12 upon lipopolysaccharide (LPS) activation. We found that although IL-4 enhanced DC IL-12p70 production, it strongly impaired IL-2 secretion by BMDCs. This inhibition, which depends on the presence of IL-4 during LPS activation, is DC specific, as IL-4 did not affect IL-2 secretion by T cells. Interestingly, inhibition of DC IL-2 production did not prevent DC priming of T lymphocytes. These results illustrate a new putative role for IL-4 on the regulation of the immune response and should help clarify the controversial reports on the effect of IL-4 on DCs.     

Ambient particulate matter directs nonclassic dendritic cell activation and a mixed TH1/TH2-like cytokine response by naive CD4+ T cells

BACKGROUND: Dendritic cells (DCs) translate environmental cues into T-cell activating signals, and are centrally involved in allergic airway inflammation. Ambient particulate matter (APM) is ubiquitous and associated with allergic diseases, but it is unknown whether APM directly activates DCs. OBJECTIVE: To study comprehensively the effects of APM on myeloid DC phenotype and function. METHODS: Development of DC was modeled using human CD34(+) progenitor cells. APM was collected from ambient outdoor air in Baltimore city. We studied the effects of APM on DC activation in vitro, compared with LPS. RESULTS: Ambient particulate matter enhanced DC expression of costimulatory receptors but suppressed the expression of both the endocytosis receptor CD206 and uptake of fluorescein isothiocyanate-conjugated dextran. The expression of the Toll-like pattern-recognition receptors Toll-like receptor 2 and Toll-like receptor 4 was also blunted. APM-exposed DCs secreted less IL-12 and IL-6 but exhibited increased secretion of IL-18 and IL-10 compared with LPS stimulation. A T(H)2-like pattern of cytokine production was seen in cocultures of APM-stimulated DCs and alloreactive naive CD4(+) T cells where the IL-13 to IFN-gamma ratio was reversed. This contrasted with the T(H)1 polarizing effects of LPS on DCs. CONCLUSION: We report for the first time that APM-exposed DCs direct a complex T(H)1/T(H)2-like pattern of T-cell activation by mechanisms that involve nonclassic activation of DCs. CLINICAL IMPLICATIONS: Inhaled APM can act directly on DCs as a danger signal to direct a proallergic pattern of innate immune activation.     

The proteolytic activity of the major dust mite allergen Der p 1 conditions dendritic cells to produce less interleukin-12: allergen-induced Th2 bias determined at the dendritic cell level

BACKGROUND: The proteolytic activity of the house dust mite allergen Der p 1 has recently been shown to bias Th cell subset development in favour of Th2. Apart from its direct effect on T cells, it is conceivable that the proteolytic activity of Der p 1 may induce the generation of dendritic cells (DCs) that favour a Th2 response. OBJECTIVE: To study the effect of the proteolytic activity of Der p 1 on DC functions; namely cell surface phenotype, IL-12 production and ability to favour a Th2 response. METHODS: We have generated immature DCs from peripheral blood monocytes, matured them with LPS in the presence of either proteolytically active or inactive Der p 1 and compared their functions using flow cytometric analysis. RESULTS: Here we demonstrate for the first time that DCs that have been matured in the presence of proteolytically active Der p 1 produce significantly less IL-12, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. The suppression of IL-12 production was due to the cleavage of CD40 by the proteolytic activity of Der p 1, hence rendering the DCs less responsive to stimulation through the CD40L-CD40 pathway. Furthermore, we demonstrate that DCs that have been matured in the presence of proteolytically active Der p 1 induce the production of significantly less IFN-gamma and more IL-4 by CD4 T cells, compared to DCs that have been matured in the presence of proteolytically inactive Der p 1. CONCLUSIONS: Collectively, our data provide compelling evidence for the role of the proteolytic activity of Der p 1 in directing DCs to induce Th2 subset development.     

IL-21 enhances SOCS gene expression and inhibits LPS-induced cytokine production in human monocyte-derived dendritic cells

Dendritic cells (DCs) play an important role in innate and adaptive immune responses. In addition to their phagocytic activity, DCs present foreign antigens to na?ve T cells and regulate the development of adaptive immune responses. Upon contact with DCs, activated T cells produce large quantities of cytokines such as interferon-gamma (IFN-gamma) and interleukin (IL)-21, which have important immunoregulatory functions. Here, we have analyzed the effect of IL-21 and IFN-gamma on lipopolysaccharide (LPS)-induced maturation and cytokine production of human monocyte-derived DCs. IL-21 and IFN-gamma receptor genes were expressed in high levels in immature DCs. Pretreatment of immature DCs with IL-21 inhibited LPS-stimulated DC maturation and expression of CD86 and human leukocyte antigen class II (HLAII). IL-21 pretreatment also dramatically reduced LPS-stimulated production of tumor necrosis factor alpha, IL-12, CC chemokine ligand 5 (CCL5), and CXC chemokine ligand 10 (CXCL10) but not that of CXCL8. In contrast, IFN-gamma had a positive feedback effect on immature DCs, and it enhanced LPS-induced DC maturation and the production of cytokines. IL-21 weakly induced the expression Toll-like receptor 4 (TLR4) and translation initiation region (TIR) domain-containing adaptor protein (TIRAP) genes, whereas the expression of TIR domain-containing adaptor-inducing IFN-beta (TRIF), myeloid differentiation (MyD88) 88 factor, or TRIF-related adaptor molecule (TRAM) genes remained unchanged. However, IL-21 strongly stimulated the expression of suppressor of cytokine signaling (SOCS)-1 and SOCS-3 genes. SOCS are known to suppress DC functions and interfere with TLR4 signaling. Our results demonstrate that IL-21, a cytokine produced by activated T cells, can directly inhibit the activation and cytokine production of myeloid DCs, providing a negative feedback loop between DCs and T lymphocytes.     

H2-D(d)-mediated upregulation of interleukin-4 production by natural killer T-cell and dendritic cell interaction

Natural killer T (NKT) cells are capable of subserving apparently opposite functions, the interferon-gamma (IFN-gamma)-mediated enhancement of host defence and interleukin-4 (IL-4) -mediated immune regulation. Although dendritic cells (DCs) potently activate NKT cells, DC regulation of the IL-4-IFN-gamma balance via NKT-cell activation is not well characterized. In the present study, we examined the effect of DC treatment with CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 ligand, on the induction of NKT-cell cytokine production. CpG-ODN-conditioned and alpha-galactosylceramide (alpha-GalCer)-loaded myeloid DCs (CpG-DCs) from BALB/c mice showed enhanced ability to induce NKT-cell production of IL-4, but not IFN-gamma, compared to alpha-GalCer-loaded control DCs (not treated with CpG-ODN). The CpG-DCs expressed significantly higher levels of H2-D(d) than control DCs, and blocking of the H2-D(d) and Ly49 receptor interaction during antigen presentation completely abolished the enhanced ability of the CpG-DCs to induce NKT-cell production of IL-4. These findings demonstrate that DC recognition of the CpG motif leads to induction of enhanced IL-4 production by NKT cells via interaction of the augmented H2-D(d) with Ly49 receptors on NKT cells.     

Flagellin enhances NK cell proliferation and activation directly and through dendritic cell-NK cell interactions

Flagellin, the principal component of bacterial flagella, is a ligand for Toll-like receptor 5 (TLR5) or TLR11 and contributes to systemic inflammation during sepsis through activation of dendritic cells (DCs) and other cells of the innate immune system. Here, we report that flagellin and the TLR4 ligand, lipopolysaccharide (LPS), induced phenotypic and functional maturation of murine bone marrow-derived DCs and enhanced DC accumulation in the draining popliteal lymph node following their footpad injection. It is interesting that flagellin injection enhanced myeloid (CD8alpha(-1)) and plasmacytoid (plasmacytoid DC antigen(+) B220(+)) DC subsets, whereas LPS only increased myeloid DCs in the draining lymph node. In addition, the footpad injection of flagellin or LPS induced significant CD4(+) T cell activation in the draining popliteal lymph node, as judged by increased CD69 or CD25 expression. We illustrate, for the first time, that flagellin also increases natural killer (NK) cell number and activation status in the draining lymph node after footpad injection. Using coculture with enriched carboxy-fluorescein diacetate succinimidyl ester-labeled NK cells, flagellin-treated DCs induce significant NK cell proliferation and activation. In fact, direct treatment of NK cells with flagellin induces a greater increase in cell proliferation than treatment with LPS. In contrast, flagellin treatment of NK cells was not a strong inducer of interferon-gamma (IFN-gamma) production, indicating that NK cell proliferation and IFN-gamma production may be regulated differentially. These data suggest that flagellin is a capable maturation agent for murine myeloid-derived DCs, and flagellin-activated DCs and flagellin itself are potent inducers of NK cell proliferation.     

Dendritic cells generated in the presence of interferon-alpha stimulate allogeneic CD4+ T-cell proliferation: modulation by autocrine IL-10, enhanced T-cell apoptosis and T regulatory type 1 cells

Dendritic cells (DCs) generated in the presence of IFN-alpha (IFN-DCs) exhibit high expression of major histocompatibility and co-stimulatory molecules and a potent ability to stimulate CD8(+) T-cell responses. Here, we found that IFN-DCs were more potent stimulators of bulk and purified CD8(+) T-cell proliferation, as compared with IL-4-DCs. In contrast, IFN-DCs were less efficient than IL-4-DCs in stimulating allogeneic CD4(+) T-cell proliferation, due to a weak induction of naive CD4(+)CD45RO(-) T-cell proliferation by these DCs. However, both DC populations induced similar levels of proliferation of memory CD4(+)CD45RO(+) T cells. IFN-DCs and IL-4-DCs exhibited a similar phenotype and production of IL-10 following maturation induced by CD40 ligation. In contrast, IFN-DCs produced higher levels of IL-10 during the first days of differentiation. In addition, neutralization of IL-10 during the differentiation of DCs increased the expression of DC-LAMP and MHC class II by IFN-DCs, and the ability of IFN-DCs to stimulate allogeneic CD4(+) T-cell proliferation at similar levels, than IL-4-DCs. Independently of IL-10 production, IFN-DCs were found to induce higher levels of CD4(+)T-cell apoptosis, this effect being more sticking on naive T cells. Finally, we demonstrated that IFN-DCs induced a differentiation bias of naive CD4(+) T cells towards Th1 and Tr1 cells, compared to IL-4-DCs. Taken together, these results indicate that, despite the induction of Tr1 cells and enhanced apoptosis of naive CD4(+) T cells, IFN-DCs are potent stimulators of CD8(+) and memory CD4(+) T cells, and induce a strong polarization of naive CD4(+) T cells towards Th1 cells, further supporting their use in immune-based therapy.     

Polarization of naive T cells into Th1 or Th2 by distinct cytokine-driven murine dendritic cell populations: implications for immunotherapy

Dendritic cells (DCs) activate T cells and regulate their differentiation into T helper cell type 1 (Th1) and/or Th2 cells. To identify DCs with differing abilities to direct Th1/Th2 cell differentiation, we cultured mouse bone marrow progenitors in granulocyte macrophage-colony stimulating factor (GM), GM + interleukin (IL)-4, or GM + IL-15 and generated three distinct DC populations. The GM + IL-4 DCs expressed high levels of CD80/CD86 and major histocompatibility complex (MHC) class II and produced low levels of IL-12p70. GM and GM + IL-15 DCs expressed low levels of CD80/CD86 and MHC class II. The GM + IL-15 DCs produced high levels of IL-12p70 and interferon (IFN)-gamma, whereas GM DCs produced only high levels of IL-12p70. Naive T cells stimulated with GM + IL-4 DCs secreted high levels of IL-4 and IL-5 in addition to IFN-gamma. In contrast, the GM + IL-15 DCs induced higher IFN-gamma production by T cells with little or no Th2 cytokines. GM DCs did not induce T cell polarization, despite producing large amounts of IL-12p70 following activation. A similar pattern of T cell activation was observed after in vivo administration of DCs. These data suggest that IL-12p70 production alone, although necessary for Th1 differentiation, is not sufficient to induce Th1 responses. These studies have implications for the use of DC-based vaccines in immunotherapy of cancer and other clinical conditions.     

Interferon-alpha2a is sufficient for promoting dendritic cell immunogenicity

Type I interferons (IFNs) are widely used therapeutically. IFN-alpha2a in particular is used as an antiviral agent, but its immunomodulatory properties are poorly understood. Dendritic cells (DCs) are the only antigen-presenting cells able to prime naive T cells and therefore play a crucial role in initiating the adaptive phase of the immune response. We studied the effects of IFN-alpha2a on DC maturation and its role in determining Th1/Th2 equilibrium. We found that IFN-alpha2a induced phenotypic maturation of DCs and increased their allostimulatory capacity. When dendritic cells were stimulated simultaneously by CD40 ligation and IFN-alpha2a, the production of interleukin (IL)-10 and IL-12 was increased. In contrast, lipopolysaccharide (LPS) stimulation in the presence of IFN-alpha2a mainly induced IL-10 release. The production of IFN-gamma and IL-5 by the responder naive T cells was also amplified in response to IFN-alpha2a-treated DCs. Furthermore, IL-12 production by IFN-alpha2a-treated DCs was enhanced further in the presence of anti-IL-10 antibody. Different results were obtained when DCs were treated simultaneously with IFN-alpha2a and other maturation factors, in particular LPS, and then stimulated by CD40 ligation 36 h later. Under these circumstances, IFN-alpha2a did not modify the DC phenotype, and the production of IL-10/IL-12 and IFN-gamma/IL-5 by DCs and by DC-stimulated naive T cells, respectively, was inhibited compared to the effects on DCs treated with maturation factors alone. Altogether, this work suggests that IFN-alpha2a in isolation is sufficient to promote DC activation, however, other concomitant events, such as exposure to LPS during a bacterial infection, can inhibit its effects. These results clarify some of the in vivo findings obtained with IFN-alpha2a and have direct implications for the design of IFN-alpha-based vaccines for immunotherapy.     

Neonatal dendritic cells are intrinsically biased against Th-1 immune responses

Dendritic cells (DCs) were derived from human peripheral blood monocytes or cord blood monocytes cultured in the presence of IL-4 and GM-CSF. Adult and cord DCs were observed to have comparable immature phenotypes. However, the increase in surface expression of HLA-DR and CD86 after addition of LPS was significantly attenuated in cord DCs, with CD25 and CD83 expression also markedly reduced. Cord DCs were also unable to produce IL-12p70, failed to down-regulate expression of the chemokine receptor CCR5 and induced lower levels of IFN-gamma production from allogeneic naive CD4+ T cells than their adult counterparts. In contrast, the kinetics of the production of TNF-alpha and IL-10 in response to LPS stimulation was comparable to adult DCs. The reduced ability of cord DCs to attain a fully mature adult phenotype, and to activate naive CD4+ T cells to produce IFN-gamma, suggests that they are intrinsically preprogrammed against the generation of Th-1 immune responses.     

Differential regulation of DC function by Siphonodiol

Siphonodiol is a polyacetylene diol isolated from marine sponges Callyspongia sp. We demonstrate that the effect of Siphonodiol on the phenotypic and functional maturation of human monocyte derived DC in vitro. Human monocytes were exposed to Siphonodiol alone, or in combination with LPS and thereafter co-cultured with na?ve T cells. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR on LPS-primed DC were partially enhanced by Siphonodiol. Siphonodiol augmented the T cell stimulatory capacity in an allo MLR to LPS-primed DC. Siphonodiol dose-dependently enhanced the production of IL-12p70 by LPS-primed DC and this cytokine production was inhibited by anti-TLR4 mAb. IFN-gamma secretion from naive T cells co-cultured with DC differentiated with LPS was augmented by Siphonodiol. These results suggest that the enhancement of Th1 cells polarization to LPS-primed DC induced by Siphonodiol depends on TLR4 and via the activation of IL-12p70.     

Adhesive substrate-modulation of adaptive immune responses

While it is well-known that adsorbed proteins on implanted biomaterials modulate inflammatory responses, modulation of dendritic cells (DCs) via adhesion-dependent signaling has only been begun to be characterized. In this work, we demonstrate that adhesive substrates elicit differential DC maturation and adaptive immune responses. We find that adhesive substrates support similar levels of DC adhesion and expression of stimulatory and co-stimulatory molecules. Conversely, DC morphology and differential production of pro- and anti-inflammatory cytokines (IL-12p40 and IL-10, respectively) is adhesive substrate-dependent. For example, DCs cultured on collagen and vitronectin substrates generate higher levels of IL-12p40, whereas DCs cultured on albumin and serum-coated tissue culture-treated substrates produce the higher levels of IL-10 compared to other substrates. Additionally, our results suggest substrate-dependent trends in DC-mediated allogeneic CD4(+) T-cell proliferation and T-helper cell type responses. Specifically, we show that substrate-dependent modulation of DC IL-12p40 cytokine production correlates with CD4(+) T-cell proliferation and T(h)1 type response in terms of IFN-gamma producing T-helper cells. Furthermore, our results suggest substrate-dependent trends in DC-mediated stimulation of IL-4 producing T-cells, but this T(h)2 type response is not dependent on DC production of IL-10 cytokine. This work has impact in the rational design of biomaterials for diverse applications such as tissue-engineered constructs, synthetic particle-based vaccines and the ex vivo culture of DCs for immunotherapies.     

Phosphatidylserine inhibits NFκB and p38 MAPK activation in human monocyte derived dendritic cells

Phosphatidylserine (PS) is an anionic phospholipid restricted to the inner surface of the plasma membrane. PS translocates to the cell surface during early apoptosis where it serves as a marker for rapid uptake by phagocytes. PS is also thought to regulate immune responses. Dendritic cells (DC) are the most potent antigen presenting cells. Previous studies demonstrated that PS inhibits the expression of MHC and co-stimulatory molecules, the secretion of IL-12p70, and the ability to activate T cells by human monocyte derived DCs. However, the cell signaling mechanisms by which PS regulated DCs are not well described. In the current study we tested the effects of PS on signal transduction pathways thought to regulate human myeloid DC maturation and IL-12p70 production. We showed that PS inhibited the activation of nuclear factor-κB (NFκB) in response to LPS by preventing IκBα phosphorylation and degradation. PS also increased the total IκBα levels in immature DCs and inhibited p38 mitogen activated protein kinase (MAPK) phosphorylation and activation. The findings suggest a possible mechanism for regulating the immunostimulatory function of DCs by PS.     

Rapid lipopolysaccharide-induced differentiation of CD14(+) monocytes into CD83(+) dendritic cells is modulated under serum-free conditions by exogenously added IFN-gamma and endogenously produced IL-10.

We showed previously that about half of purified CD14(+) peripheral blood monocytes cultured under serum-free conditions and treated with GM-CSF and bacterial LPS rapidly (2 - 4 day) differentiate into CD83(+) dendritic cells (DC). The remaining cells retain the CD14(+)/CD83(-) monocyte/macrophage phenotype. In order to identify factors that influence whether monocytes differentiate into DC or remain on the monocyte/macrophage developmental pathway, we evaluated the effects of exogenously added IFN-gamma and endogenously produced IL-10 on the proportion and function of CD14(+) monocytes that adopt DC characteristics in response to LPS. IFN-gamma priming dramatically increased the proportion of monocytes that adopted stable DC characteristics in response to LPS, improved their T cell allosensitizing capacity, and enhanced levels of secreted IL-12 heterodimer. IFN-gamma priming also suppressed the production of IL-10, a cytokine known to have inhibitory effects on DC differentiation. When monocytes were treated with LPS plus IL-10-neutralizing antibodies, dramatically enhanced DC differentiation, IL-12 secretion, and T cell allosensitizing capacity were observed, mimicking in many respects the effects of IFN-gamma priming. IFN-gamma primed cells still displayed appreciable sensitivity to exogenously added IL-10, suggesting that attenuated IL-10 secretion is partially responsible for the enhancing effects of IFN-gamma. These studies therefore identify IFN-gamma as a DC differentiation co-factor for CD14(+) monocytes, and IL-10 as an autocrine/paracrine inhibitor of DC differentiation, linking these agents for the first time as mutually opposed regulators that govern whether CD14(+) cells differentiate into DC upon contact with LPS or remain on the monocyte/macrophage developmental pathway.     

Sphingosine kinase inhibitor suppresses a Th1 polarization via the inhibition of immunostimulatory activity in murine bone marrow-derived dendritic cells

Sphingosine kinase (Sphk) has been shown to be activated by growth factor and survival factors, and one of its products, sphingosine-1-phosphate, plays an important role in the regulation of various cellular responses. However, the effect of Sphk on the maturation and immunostimulatory function of dendritic cells (DCs) still remains largely unknown. In this study, we examined whether sphingosine kinase inhibitor (SKI) can influence co-stimulatory molecules (CD40, CD80, CD86 and MHC class II) and cytokine production (IL-12 and IL-10) in murine bone marrow-derived DCs. SKI significantly inhibited co-stimulatory molecules in DCs. SKI suppressed IL-12 production by DCs and IFN-gamma production by T cells. In addition, SKI-inhibited LPS induced the translocation of nuclear factor-kappaB, whereas it did not affect the degradation of IL-1 receptor-associated kinase-1 by LPS. These novel findings provide new insight into the immunopharmacological role of SKI in terms of its effects on DCs. These findings open a possibility for further understanding of the immunopharmacological functions of SKI, as well as therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.