moDC Maturation Research Articles

Effects of TLR Agonists on the Hypoxia-Regulated Transcription Factor HIF-1α and Dendritic Cell Maturation under Normoxic Conditions

Dendritic cells (DC) are professional antigen presenting cells that represent an important link between innate and adaptive immunity. Danger signals such as toll-like receptor (TLR) agonists induce maturation of DC leading to a T-cell mediated adaptive immune response. In this study, we show that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induce the expression of HIF-1α in human monocyte-derived DC under normoxic conditions. On the functional level, inhibition of HIF-1α using chetomin (CTM), YC-1 and digoxin lead to no consistent effect on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1α stabilization or activity through different mechanisms. Stabilization of HIF-1α protein by hypoxia or CoCl2 did not result in maturation of human DC. In addition, we could show that TLR stimulation resulted in an increase of HIF-1α controlled VEGF secretion. These results show that stimulation of human MoDC with exogenous as well as endogenous TLR agonists induces the expression of HIF-1α in a time-dependent manner. Hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Current evidence suggests that different target genes may be affected by HIF-1α under normoxic conditions with physiological roles that differ from those induced by hypoxia.

Human defensins activate monocyte-derived dendritic cells, promote the production of proinflammatory cytokines, and up-regulate the surface expression of CD91

Defensins are endogenous defense peptides with well defined antimicrobial activity against a broad spectrum of pathogens including bacteria, fungi, viruses, and parasites.Several lines of evidence suggest that defensins might also contribute to the regulation of host innate and adaptive immunity, but their immunomodulatory functions are still poorly understood. Herein, we studied the impact of human defensins on multiple functions of DCs, which are a central player in all immune responses, bridging innate and adaptive immunity. We challenged DCs differentiated in vitro from human moDCs with HNP-1 alpha-defensin or HBD-1. HNP-1 and HBD-1 were chemotactic for moDCs. Both defensins promoted the activation and maturation of moDCs, as assessed by up-regulation of surface expression of the costimulatory molecules CD80, CD86, and CD40, the maturation marker CD83, and HLA-DR. HNP-1 and HBD-1 also enhanced the production of the proinflammatory cytokines TNF-alpha, IL-6, and IL-12p70 but did not affect the production of the regulatory cytokine IL-10. According to these stimulatory effects, HNP-1 and HBD-1 increased the allostimulatory activity of moDCs significantly. Finally, HNP-1 and HBD-1 promoted the up-regulation of CD91 on the DC surface. CD91 is a scavenger receptor involved in the recognition of multiple ligands including defensins, thus suggesting that defensins may amplify their own effects through the activation of an autocrine loop. Taken together, our observations may provide new insight into the immunomodulatory properties of human defensins and may aid the exploration of new therapeutic strategies to potentiate antimicrobial and antitumor immunity.

Impact of Psoralen/UVA-Treatment on Survival, Activation, and Immunostimulatory Capacity of Monocyte-Derived Dendritic Cells

Extracorporeal Photopheresis (ECP) has been shown to be an effective treatment of graft-versus-host disease, solid organ graft rejection, and other T-cell-mediated diseases. The mechanisms of action of ECP include lymphocyte apoptosis, cytokine modulation, and the induction of regulatory T cells. It has been suggested that dendritic cells (DCs) are more resistant to ECP-induced apoptosis and might be directly modulated by ECP. We tested this hypothesis using in vitro Psoralen/UVA (PUVA) treatment as an in vitro model of ECP. Monocyte-derived DCs (mo-DCs) were treated with 8-methoxypsoralen /UVA and analyzed for surface molecule expression, apoptosis markers, endocytosis, and migratory and immunostimulatory capacity. Mo-DC phenotype and cytokine secretion was tested after CD40L stimulation. Naive T cells stimulated with PUVA-treated mo-DCs were tested for Th1/Th2 cytokine secretion and associated chemokine receptor patterns. DCs underwent apoptosis after in vitro PUVA and in vivo ECP. In vitro, the induction of apoptosis was preceded by partial maturation of immature mo-DCs. PUVA-treated immature mo-DCs also exhibited enhanced migratory and immunostimulatory capacity. However, mo-DCs stimulation through CD40 ligation was abrogated and interleukin (IL)-12 secretion was abolished 24 hr after PUVA treatment. PUVA-treated mo-DCs skewed naive T cells toward a Th2 response as defined by increased IL-4, IL-10, and IL-13 and decreased interferon-gamma levels, and the expression of the Th2-associated chemokine receptors CCR4 and CCR10. The observed Th2 shift was partially reversed by exogenous IL-12. These data suggest that direct modulation of DC function as well as apoptosis contribute to the immunoregulatory effects of ECP.

Potentially probiotic bacteria induce efficient maturation but differential cytokine production in human monocyte-derived dendritic cells

To analyze the ability of nine different potentially probiotic bacteria to induce maturation and cytokine production in human monocyte-derived dendritic cells (moDCs). Cytokine production and maturation of moDCs in response to bacterial stimulation was analyzed with enzyme-linked immunosorbent assay (ELISA) and flow cytometric analysis (FACS), respectively. The kinetics of mRNA expression of cytokine genes was determined by Northern blotting. The involvement of different signaling pathways in cytokine gene expression was studied using specific pharmacological signaling inhibitors. All studied bacteria induced the maturation of moDCs in a dose-dependent manner. More detailed analysis with S. thermophilus THS, B. breve Bb99, and L. lactis subsp. cremoris ARH74 indicated that these bacteria induced the expression of moDC maturation markers HLA class II and CD86 as efficiently as pathogenic bacteria. However, these bacteria differed in their ability to induce moDC cytokine gene expression. S. thermophilus induced the expression of pro-inflammatory (TNF-alpha, IL-12, IL-6, and CCL20) and Th1 type (IL-12 and IFN-gamma) cytokines, while B. breve and L. lactis were also potent inducers of anti-inflammatory IL-10. Mitogen-activated protein kinase (MAPK) p38, phosphatidylinositol 3 (PI3) kinase, and nuclear factor-kappa B (NF-kappaB) signaling pathways were shown to be involved in bacteria-induced cytokine production. Our results indicate that potentially probiotic bacteria are able to induce moDC maturation, but their ability to induce cytokine gene expression varies significantly from one bacterial strain to another.

Surface α2-3- and α2-6-sialylation of human monocytes and derived dendritic cells and its influence on endocytosis

Several glycoconjugates are involved in the immune response. Sialic acid is frequently the glycan terminal sugar and it may modulate immune interactions. Dendritic cells (DCs) are antigen-presenting cells with high endocytic capacity and a central role in immune regulation. On this basis, DCs derived from monocytes (mo-DC) are utilised in immunotherapy, though many features are ignored and their use is still limited. We analyzed the surface sialylated glycans expressed during human mo-DC generation. This was monitored by lectin binding and analysis of sialyltransferases (ST) at the mRNA level and by specific enzymatic assays. We showed that alpha 2-3-sialylated O-glycans and alpha 2-6- and alpha 2-3-sialylated N-glycans are present in monocytes and their expression increases during mo-DC differentiation. Three main ST genes are committed with this rearrangement: ST6Gal1 is specifically involved in the augmented alpha 2-6-sialylated N-glycans; ST3Gal1 contributes for the alpha2-3-sialylation of O-glycans, particularly T antigens; and ST3Gal4 may contribute for the increased alpha2-3-sialylated N-glycans. Upon mo-DC maturation, ST6Gal1 and ST3Gal4 are downregulated and ST3Gal1 is altered in a stimulus-dependent manner. We also observed that removing surface sialic acid of immature mo-DC by neuraminidase significantly decreased its endocytic capacity, while it increased in monocytes. Our results indicate the STs expression modulates the increased expression of surface sialylated structures during mo-DC generation, which is probably related with changes in cell mechanisms. The ST downregulation after mo-DC maturation probably results in a decreased sialylation or sialylated glycoconjugates involved in the endocytosis, contributing to the downregulation of one or more antigen-uptake mechanisms specific of mo-DC.

The B7 Homolog Butyrophilin BTN2A1 Is a Novel Ligand for DC-SIGN

The MHC-encoded butyrophilin, BTN2A1, is a cell surface glycoprotein related to the extended family of B7 costimulatory molecules. BTN2A1 mRNA was expressed in most human tissues, but protein expression was significantly lower in leukocytes. An Ig-fusion protein of BTN2A1 bound to immature monocyte-derived dendritic cells. Binding diminished upon MoDC maturation and no binding was detected to Langerhans cells. Induction of the counterreceptor was IL-4 dependent and occurred early during dendritic cell differentiation. The interaction required the presence of Ca2+ and was mediated by high-mannose oligosaccharides. These properties matched DC-SIGN, a DC-specific HIV-1 entry receptor. This was confirmed by binding of soluble BTN2A1 to DC-SIGN-transfectants and its inhibition by a specific Ab. DC-SIGN bound to native BTN2A1 expressed on a range of tissues. However, BTN2A1 was not recognized on some normal cells such as HUVECs despite a similar expression level. The BTN2A1 of tumor cells such as HEK293T have more high-mannose moieties in comparison to HUVECs, and those high-mannose moieties are instrumental for binding to DC-SIGN. The data are consistent with tumor- or tissue-specific glycosylation of BTN2A1 governing recognition by DC-SIGN on immature monocyte-derived dendritic cells.

Neutrophil Elastase Converts Human Immature Dendritic Cells into Transforming Growth Factor-β1-Secreting Cells and Reduces Allostimulatory Ability

During microbial infection, neutrophils (polymorphonuclear leukocytes; PMNs) activate dendritic cells (DCs). However, early reports illustrated that neutrophil-derived mediators may suppress responses to mitogens. In the present study, we investigated the mechanism used by PMNs to modulate the immunostimulatory ability of DCs. Autologous syngeneic PMNs decreased T-cell proliferation induced by allogeneic DCs. Culture supernatant (CS) derived from PMNs also decreased allostimulation ability of immature DCs and increased the expression of transforming growth factor (TGF)-beta1 on DCs. A TGF-beta1 monoclonal antibody, a CD40 monoclonal antibody, or a serine protease inhibitor reversed the effect of PMN CS on DC allostimulatory ability. Furthermore, elastase reproduced the inhibitory effect of PMN CS on DC allostimulatory ability and the TGF-beta1 production. The role of elastase was confirmed by examining PMN CS from two patients with cyclic neutropenia, a disease due to mutations in the neutrophil elastase gene. These PMN CS samples had reduced elastase activity and were unable to increase DC TGF-beta1 production. Moreover, elastase and PMN CS induced IkappaBalpha degradation in DCs. We conclude that PMNs decrease DC allostimulatory ability via production of elastase leading to a switch of immature DCs into TGF-beta1-secreting cells.

TLR-4 and -6 agonists reverse apoptosis and promote maturation of simian virus 5-infected human dendritic cells through NFkB-dependent pathways

Infection of primary cultures of human immature monocyte-derived dendritic cells (moDC) with the paramyxovirus Simian Virus 5 (SV5) results in extensive cytopathic effect (CPE) and induction of apoptosis, but DC maturation pathways are not activated. In this study, we investigated the relationship between SV5-induced apoptosis and the lack of DC maturation. Reducing CPE and apoptosis in SV5-infected immature DC by the addition of a pancaspase inhibitor resulted in only low level expression of maturation markers CD40, CD80 and CD86, suggesting that SV5 infection either actively blocked maturation pathways or failed to provide sufficient signals to activate maturation. To distinguish between these hypotheses, SV5-infected immature DC were challenged with agonists that stimulate toll-like receptors (TLRs). Treatment with the TLR-4 agonist LPS or TLR-6 agonist FSL1 enhanced cell surface expression of CD40, CD80 and CD86 on SV5-infected cells to levels approaching that of mock-infected TLR-treated moDC, but treatment with agonists for TLR-2, -3, -5 or -8 had little effect. Addition of TLR-4 or -6 agonists to SV5-infected DC also dramatically reduced CPE and apoptosis, but the levels of viral protein and virus yield were not affected. Similarly, SV5-infected immature moDC were matured by treatment with IL-1β, and these mature infected cells also showed reduced CPE and apoptosis. In the presence of NFkB inhibitors, TLR-4 and -6 agonists did not promote maturation or reduce apoptosis of SV5-infected DC, indicating that maturation and cell survival were both dependent on signaling through NFkB-dependent pathways. Our results suggest a model whereby SV5 replication induces apoptosis in immature DC but fails to provide strong maturation signals, while activation of NFkB-dependent pathways by exogenous ligands can lead to moDC maturation and override SV5-induced cell death.

Inhibitory effects of suplatast tosilate on the differentiation and function of monocyte‐derived dendritic cells from patients with asthma

Dendritic cells (DCs) are antigen-presenting cells that efficiently activate T cells. We examined the effects of suplatast tosilate, which prevents T-helper type 2 responses, on the differentiation and function of monocyte-derived DCs (moDCs). DCs were differentiated in vitro from peripheral monocytes from patients with asthma by the addition of granulocyte macrophage colony-stimulating factor and IL-4 in the presence or absence of suplatast tosilate. Cell surface molecules (CD1a, CD14, CD80, CD83, CD86, HLA-DR) on immature and mature DCs were analysed with flow cytometry, and the secretion of CC chemokine ligand (CCL)17 (thymus and activation-regulated chemokine), IL-12p70, IL-12p40, and IL-10 was measured with an ELISA. We also studied the proliferative responses of allogeneic CD4(+) T cells from healthy subjects to DCs differentiated in the presence of suplatast tosilate. In addition, the production of IFN-gamma and IL-5 by CD4(+) T cells after coculture with untreated DCs or suplatast tosilate-treated DCs was measured with ELISA. Suplatast tosilate significantly inhibited the expression of CD1a, CD80, and CD86 on immature DCs and of CD1a, CD80, CD83, and CD86 on mature DCs. Suplatast tosilate also significantly inhibited the secretion of CCL17, IL-12p70, and IL-12p40; however, the secretion of IL-10 was not affected. The proliferative responses of allogeneic CD4(+) T cells to suplatast tosilate-treated DCs were suppressed. Moreover, suplatast tosilate-treated DCs had an impaired capacity to stimulate CD4(+) T cells to produce IFN-gamma and IL-5. Suplatast tosilate inhibits the differentiation, maturation, and function of moDCs.

Antitumor action and immune activation through cooperation of bee venom secretory phospholipase A2 and phosphatidylinositol-(3,4)-bisphosphate.

We evaluated tumor cell growth modulation by bee venom secretory phospholipase A2 (bv-sPLA2) and phosphatidylinositol-(3,4)-bisphosphate as well as potential cooperative effects. In addition, the immunomodulatory impact of tumor cell treatment was examined by monitoring changes in phenotype and function of monocyte-derived dendritic cells (moDCs) cocultured with pretreated tumor cells. Bv-sPLA2 or phosphatidylinositol-(3,4)-bisphosphate alone displayed moderate effects on the proliferation of A498 renal cell carcinoma cells, T-47D breast cancer cells, DU145 prostate cancer cells and BEAS-2B transformed lung cells. However, when bv-sPLA2 was coadministered with phosphatidylinositol-(3,4)-bisphosphate a potent inhibition of [3H] thymidine incorporation into all tested cell lines occurred. This inhibition was due to massive cell lysis that reduced the number of cells with proliferative capacity. Importantly, tumor cell lysates generated with bv-sPLA2 plus phosphatidylinositol-(3,4)-bisphosphate induced maturation of human moDCs demonstrated by enhanced expression of CD83 and improved stimulation in allogeneic mixed leukocyte reactions. Our data demonstrate that bv-sPLA2 and phosphatidylinositol-(3,4)-bisphosphate synergistically generate tumor lysates which enhance the maturation of immunostimulatory human monocyte-derived dendritic cells. Such tumor lysates which represent complex mixtures of tumor antigens and simultaneously display potent adjuvant properties meet all requirements of a tumor vaccine.

Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector

DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms. Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated. The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.

Differential Response of Dendritic Cells to Human Metapneumovirus and Respiratory Syncytial Virus

Dendritic cells (DCs) play a pivotal role in shaping antiviral immune responses in the respiratory tract. Human metapneumovirus (hMPV) is a recently identified pathogen and like its better known relative, respiratory syncytial virus (RSV), has been increasingly recognized as a major cause of respiratory morbidity in infants and in elderly persons. In the present study, we examined susceptibility as well as cellular responses of human DCs to hMPV compared with RSV. Monocyte-derived DCs (moDCs) were susceptible to infection by both viruses, but only RSV was able to induce a productive infection with release of viral progeny. Despite the fact that viral infection resulted in phenotypic maturation of moDCs, as shown by the upregulation of cell surface markers and antigen-presenting molecules (MHC I and II, CD80, CD83, CD86, CD38), RSV-infected moDCs showed a severely impaired capacity to stimulate CD4+ T cell proliferation. Compared with hMPV, RSV was a more potent inducer of inflammatory and immunomodulatory cytokines, including TNF-alpha, IL-6, IL-1beta, IL-10, and IL-12p70 in both moDCs and plasmacytoid dendritic cells (pDCs). On the other hand, hMPV, but not RSV, was able to trigger production of IFN-alpha by moDCs, while both viruses strongly induced IFN-alpha in pDCs. Finally, both viruses strikingly suppressed IFN-alpha production by moDCs or pDCs stimulated with synthetic dsRNA and CpG-ODN, respectively. The findings provide novel evidence that RSV and hMPV differentially activate human DCs and may use distinct mechanisms to interfere with the host innate and adaptive immune responses.

Dendritic cell maturation by CD11c− T cells and Vα24+ natural killer T‐cell activation by α‐Galactosylceramide

Human invariant Valpha24+ natural killer T (NKT) cells display potent antitumor activity upon stimulation. Activation of endogenous Valpha24+ NKT cells would be one strategy for the treatment of cancer patients. For example, dendritic cells (DCs) loaded with a glycolipid NKT cell ligand, alpha-galactosylceramide (alphaGalCer, KRN7000), are a possible tool for the activation and expansion of functional Valpha24+ NKT cells in vivo. In this report, we demonstrate that the levels of expansion and the ability to produce IFN-gamma of Valpha24+ NKT cells induced by alphaGalCer-loaded whole PBMCs cultured with IL-2 and GM-CSF (IL-2/GM-CSF-cultured PBMCs) were superior to those of cells induced by monocyte-derived CD11c+ DCs (moDCs) developed with IL-4 and GM-CSF. Interestingly, CD11c+ cells in the IL-2/GM-CSF-cultured PBMCs showed a mature phenotype without further stimulation and exerted potent stimulatory activity on Valpha24+ NKT cells to enable them to produce IFN-gamma preferentially at an extent equivalent to mature moDCs induced by stimulation with LPS or a cytokine cocktail. Cocultivation with CD11c- cells in the IL-2/GM-CSF-cultured PBMCs induced maturation of moDCs. In particular, CD11c-CD3+ T cells appeared to play important roles in DC maturation. In addition, TNF-alpha was preferentially produced by CD11c-CD3+ T cells in IL-2/GM-CSF-cultured PBMCs and was involved in the maturation of moDCs. Thus, the maturation of DCs induced by CD11c- T cells through TNF-alpha production appears to result in the efficient expansion and activation of Valpha24+ NKT cells to produce IFN-gamma preferentially.

Triptolide affects the differentiation, maturation and function of human dendritic cells

Triptolide is a purified component from a traditional Chinese herb Tripterygium wilfordii Hook F. It has been shown to have anti-inflammatory and immunosuppressive activities by its inhibitory effect on T cells. But the effect of triptolide on dendritic cells (DC) is unknown. Dexamethasone (Dex) is a classic immunosuppressive agent known to suppress the immune response at different levels and has recently found to modulate the development of DC, thereby influencing the initiation of the immune response. In this study, we investigated the affect of triptolide on the differentiation, maturation and function of DC differentiated from human monocytes (MoDC) in vitro in the presence of GM-CSF and IL-4. Dex was included in the study as a reference. Our data show that both triptolide and Dex prevented the differentiation in immature MoDC by inhibiting CD1a, CD40, CD80, CD86 and HLA-DR expression but upregulating CD14 expression, as well as by reducing the capacity of MoDC to stimulate lymphocyte proliferation in the allogeneic mixed lymphocyte reaction. They blocked the maturation of MoDC as totally blocked induction of CD83 expression and absent upregulation of CD40, CD80, CD86 and HLA-DR. In addition, higher concentration of triptolide (20 ng/ml) and 10 − 6 M Dex induced apoptosis in MoDC as measured by expression of APO2.7 and DNA fragmentation (TUNEL assay). However, the phagocytic capacity of MoDC was enhanced by triptolide but not Dex. Therefore, the suppression of DC differentiation, the function in immature DCs as well as the inhibition of DC maturation by triptolide may explain some of its immunosuppressive properties. It is suggested that DCs are a primary target of the immunosuppressive activity of triptolide.

Interleukin 10 Induces the Maturation of Putative Tolerogenic Monocyte-derived Dendritic Cells as Revealed by GeneArray Analysis

IL-10 is a multifunctional cytokine with known inhibitory effect on DC maturation. In the present study, we show that the presence of IL-10 during the first 6 days of maturation of Mo-DC has severe effects on the phenotype with IL-10 treated Mo-DC retaining a high expression of factors involved in antigen uptake and having low expression of costimulatory factors and MHC II. The effect of IL-10 on DC maturation is further characterized by a comprehensive investigation of the expression profile of 12500 genes using GeneArrays and comparing the data for IL-10 treated and untreated Mo-DC at day 6 and 8 with monocytes and macrophages. The data is confirmed with semi-quantitative RT-PCR. We find that the transcriptional pattern of IL-10 treated Mo-DC at day 6 and day 8 is distinct from both untreated Mo-DC and macrophages. IL-10 induces the expression of genes coding for CCL18, ILT3, ILT4 and TGFβ while inhibiting the expression of IL-12 p40 mRNA, and genes coding for CCL17, CCL19 and CCL22 suggesting a likely effect of IL-10 on Mo-DC maturation to be the generation of tolerogenic Mo-DC.

The cyclopentenone prostaglandin PGA2 costimulates the maturation of human dendritic cells

Dendritic cells (DCs), also referred to as the sentinels of the immune system, induce and coordinate important functions of immune surveillance. Prostaglandin E2 (PGE2), a member of the eicosanoid family of arachidonic acid derivatives, is widely used to enhance the TNF-alpha-driven maturation of human monocyte-derived DCs (moDCs) both in basic research and in clinical settings. However, PGE2 is known to rapidly undergo nonenzymatic dehydration to produce PGA2, a member of the cyclopentenone PGs, which have been implicated in anti-inflammatory processes. In a side-by-side analysis we therefore compared the influence of PGE2 and PGA2 on the TNF-alpha-induced maturation of human moDCs. Phenotypic changes, migratory responses towards MIP-3beta, and T-cell responses induced by the differentially matured moDCs were assessed. We found that PGA2 is nearly as potent as PGE2 in costimulating the TNF-alpha-induced phenotypic maturation of human moDCs. Both PGE2 and PGA2 further enhanced the migratory and T-cell-stimulatory capacity of TNF-alpha-treated moDCs. Maturation of moDCs with either PGE2 or PGA2 resulted in enhanced IFN-gamma, TNF-alpha, and IL-5 production and repressed IL-10 production in allogeneic mixed leukocyte cultures. PGE2 was always more potent than PGA2. Our data suggest that some of the effects attributed to PGE2 may in fact be mediated by its degradation product PGA2. This work also demonstrates that cyclopentenone PGs may have pro-inflammatory properties and that both PGE2 and PGA2 can contribute to the development of Th1-type immune responses.

Choosing a Dendritic Cell Profession

Circulating monocytes can differentiate into two types of professional antigen-presenting cells: macrophages or dendritic cells (DCs). However, the factors that determine which path they follow and that govern further differentiation remain incompletely understood. Bayry et al. found that DCs derived from monocytes (mo-DCs) of people with X-linked agammaglobulinemia (XLA), who have reduced numbers of circulating B cells and thus reduced amounts of circulating immunoglobulin, showed reduced expression of DC differentiation markers compared with mo-DCs from healthy donors. Stimulation with CD40 ligand or a monoclonal antibody against CD40 promoted XLA mo-DC maturation, as did exposure to physiological levels of immunoglobulin. Moreover, CD40-reactive natural antibodies (NAbs, which are produced without stimulation by foreign antigen and are often autoreactive) isolated from immunoglobulin promoted maturation of mo-DCs from both healthy donors and individuals with XLA. However, XLA mo-DCs differentiated with CD40-reactive NAbs secreted IL-10 (which limits inflammatory responses) and showed increased immunoreactivity for cAMP response element-binding protein 1 (CREB-1), whereas XLA mo-DCs stimulated with CD40 ligand secreted interleukin-12 (IL-12, which promotes inflammatory responses) and had increased immunoreactivity for NF-κB p65, NF-κB p50, and c-Rel. Thus, the authors propose that NAbs found circulating in immunoglobulin play a role in promoting the maturation of "bystander" mo-DCs, without directly promoting their ability to stimulate an immune response. J. Bayry, S. Lacroix-Desmazes, V. Donkova-Petrini, C. Carboneil, N. Misra, Y. Lepelletier, S. Delignat, S. Varambally, E. Oksenhendler, Y. Lévy, M. Debré, M. Kazatchkine, O. Hermine, S. V. Kaveri, Natural antibodies sustain differentiation and maturation of human dendritic cells. Proc. Natl. Acad. Sci. U.S.A. 101 , 14210-14215 (2004). [Abstract] [Full Text]

Activation of human dendritic cells by the PorA protein of Neisseria meningitidis.

The major porin proteins present in the outer membrane of Neisseria meningitidis, the causative agent of life-threatening meningitis and septicaemia, are believed to have potent immunostimulatory effects. In this study, the interactions between human monocyte-derived dendritic cells (mo-DC) and the PorA porin were investigated, in order to reveal the role of this protein in promoting innate and adaptive immune responses. Recombinant (r)PorA induced mo-DC maturation, as reflected by reduced receptor-mediated endocytosis, increased production of the chemokines IL-8, RANTES, MIP-1 alpha and MIP-1 beta and augmented expression of the surface markers CD40, CD54, CD80, CD86 and major histocompatibility complex class II molecules. However, rPorA induced either low level or no significant secretion of pro-inflammatory cytokines from mo-DC. The protein potently augmented the capacity of mo-DC to activate both allogeneic CD4(+) memory T-cells and CD4(+)RA(+) naïve T-cells. In addition, rPorA appeared to inhibit the production of IL-12p70 that follows from the interaction between CD40 on the mo-DC and CD40-ligand on T-cells, thereby directing T-cell differentiation towards a Th2 type response. These data demonstrate that PorA is involved in DC activation and in influencing the nature of the T-helper immune response, which are important properties for generating antibody responses required for protective immunity against meningococci and for determining the immuno-adjuvant effects of this protein.

FS13.7 CXCL8: a potential novel marker in predicting contact sensitisers

Cultured monocyte‐derived dendritic cells (moDC) have been shown to provide a promising alternative for identifying potential sensitisers in vitro. Allergen induced moDC maturation results in elevated expression of the cell surface markers CD83 and CD86. Unfortunately, this increase in CD83 and CD86 expression is not sensitive enough to identify moderate and weak contact sensitisers. In this study, we investigated whether chemokine production by moDC is increased upon maturation and, ultimately, whether chemokine production can be used as a sensitive method to discriminate allergens from irritants.Methods: MoDC were exposed for 48 hours to allergens (NiSO4, CoCl2, PdCl2, CuSO4, CrCl3, K2Cr2O7, PPDA and DNCB) and irritants (SDS, DMSO, BCl2 and propan‐1‐ol). CD83 and CD86 expression was analysed by flow cytometric determination and chemokine production (CXCL8, CCL5, CCL17 and CCL20) was determined by ELISA.Results: Exposure to strong allergens caused up‐regulation of CD83 and CD86 expression whereas exposure to irritants did not. Remarkably, increased CXCL8 production by moDC was detected after allergen exposure whereas a decrease in CXCL8 production was observed after irritant exposure. CCL20 production was induced only by NiSO4. CCL5 and CCL17 production was increased upon exposure to both allergens and irritants.Conclusion: CXCL8 production by moDC can be used to distinguish allergens from irritants. This chemokine is a potentially novel marker for determining the sensitising capacity of a chemical in vitro. Furthermore, our results suggest an important role for CXCL8 in the sensitization phase of ACD.

Activation of human dendritic cells is modulated by components of the outer membranes of Neisseria meningitidis.

Neisseria meningitidis serogroup B is a major cause of life-threatening meningitis and septicemia worldwide, and no effective vaccine is available. Initiation of innate and acquired immune responses to N. meningitidis is likely to be dependent on cellular responses of dendritic cells (DC) to antigens present in the outer membrane (OM) of the meningococcus. In this study, the responses of human monocyte-derived DC (mo-DC) to OM isolated from parent (lipopolysaccharide [LPS]-replete) meningococci and from a mutant deficient in LPS were investigated. Parent OM selectively up-regulated Toll-like receptor 4 (TLR4) mRNA expression and induced mo-DC maturation, as reflected by increased production of chemokines, proinflammatory cytokines, and CD83, CD80, CD86, CD40, and major histocompatibility complex (MHC) class II molecules. In contrast, LPS-deficient OM selectively up-regulated TLR2 mRNA expression and induced moderate increases in both cytokine production and expression of CD86 and MHC class II molecules. Preexposure to OM, with or without LPS, augmented the allostimulatory properties of mo-DC, which induced proliferation of naive CD4+ CD45RA+ T cells. In addition, LPS-replete OM induced a greater gamma interferon/interleukin-13 ratio in naive T cells, whereas LPS-deficient OM induced the reverse profile. These data demonstrate that components of the OM, other than LPS, are also likely to be involved in determining the levels of DC activation and the nature of the T-helper immune response.