Peripheral Blood Monocyte-derived Dendritic Cells Research Articles

Toll-like receptor 1 N248S polymorphism affects T helper 1 cytokine production and is associated with serum immunoglobulin E levels in Taiwanese allergic patients.

This study was carried out to investigate whether toll-like receptor-1 (TLR1) rs4833095 (N248S) variant, common in the Taiwanese population, contributes to pathogenesis of allergy. TLR2/1 ligand Pam3CSK4-stimulated cytokine production in peripheral blood mononuclear cells and monocyte-derived dendritic cells of different genotypes were measured via enzyme-linked immunosorbent assay. Ninety-three Taiwanese allergic patients (with asthma, allergic rhinitis, or atopic dermatitis) and 76 controls were recruited for genotyping. Serum immunoglobulin E (IgE) levels were evaluated in 60 allergic patients. The homozygous TLR1 C variant allele carrier had increased Pam3CSK4-induced tumor necrosis factor-α and interleukin-12 responses in peripheral blood mononuclear cells and monocyte-derived dendritic cells, respectively. Furthermore, although the C/C genotype was not associated with susceptibility to atopic diseases, it was correlated with lower total IgE levels in sera of allergic patients. Our data suggest that the TLR1 N248S polymorphism might play a role in Th1/Th2 differentiation, and the determination of serum IgE levels. However, interactions with other genetic and environmental factors might be required to contribute to risk of allergic diseases in our population.

Timing and intensity of exposure to interferon‐γ critically determines the function of monocyte‐derived dendritic cells

A growing body of evidence suggests that inflammatory cytokines have a dualistic role in immunity. In this study, we sought to determine the direct effects of interferon-γ (IFN-γ) on the differentiation and maturation of human peripheral blood monocyte-derived dendritic cells (moDC). Here, we report that following differentiation of monocytes into moDC with granulocyte-macrophage colony-stimulating factor and interleukin-4, IFN-γ induces moDC maturation and up-regulates the co-stimulatory markers CD80/CD86/CD95 and MHC Class I, enabling moDC to effectively generate antigen-specific CD4(+) and CD8(+) T-cell responses for multiple viral and tumour antigens. Early exposure of monocytes to high concentrations of IFN-γ during differentiation promotes the formation of macrophages. However, under low concentrations of IFN-γ, monocytes continue to differentiate into dendritic cells possessing a unique gene-expression profile, resulting in impairments in subsequent maturation by IFN-γ or lipopolysaccharide and an inability to generate effective antigen-specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate that IFN-γ imparts differential programmes on moDC that shape the antigen-specific T-cell responses they induce. Timing and intensity of exposure to IFN-γ can therefore determine the functional capacity of moDC.

Production of corticotropin-releasing factor and urocortin from human monocyte-derived dendritic cells is stimulated by commensal bacteria in intestine.

To examine whether commensal bacteria are a contributing cause of stress-related mucosal inflammation. Human peripheral blood monocyte-derived dendritic cells (MoDCs) were stimulated by commensal bacterial strains, including Escherichia coli, Clostridium clostridioforme, Bacteroides vulgatus (B. vulgatus), Fusobacterium varium (F. varium), and Lactobacillus delbrueckii subsp. bulgaricus. After incubation, corticotropin-releasing factor (CRF) and urocortin 1 (UCN1) mRNA in the cells was examined by real-time reverse transcription polymerase chain reaction. Supernatants from the cells were tested for CRF and UCN1 using an enzyme-linked immunosorbent assay. Both CRF and UCN1 were significantly augmented by B. vulgatus and F. varium at both the mRNA and protein levels. In particular, B. vulgatus stimulated human MoDCs, resulting in extremely high levels of CRF and UCN1. Stimulation of MoDCs by B. vulgatus and F. varium may be associated with CRF/UCN1-related intestinal disorders, such as irritable bowel syndrome and inflammatory bowel disease.

Mechanism of MBL inhibiting the LPS-induced DC maturation

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.

Development and evaluation of an in vitro skin sensitization assay based on primary monocyte-derived dendritic cells (P6376)

Abstract Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Until now, animal experiments (e.g. the local lymph node assay) are supplying most of the data used to assess the sensitization potential of new chemicals. However, becoming effective in March 2013, the 7th amendment to the EU Cosmetic Directive introduced an animal testing ban for the endpoint skin sensitisation for cosmetic ingredients. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. We successfully solved the issues associated with the use of primary cells (optimized cytokine concentrations, incubation time, readout, pooled vs. single donors and cytoxicity) resulting in a stable and reproducible protocol. Here, we report on the adaption of the protocol to the 96-well format, allowing for a higher and cost-effective throughput of test substances. Comparative analyses of the effect of several skin sensitizers on CD86 expression display similar results for 24- and 96-well format. Upscaling of the PBMDCs advances its applicability for the reliable detection of human skin sensitizers.

Roles of interleukin-10 differentiated dendritic cell of allergic asthma patients in T-lymphocyte proliferation in vitro

To explore the roles of interleukin (IL)-10 differentiated peripheral blood monocyte-derived dendritic cell (DC-10) of allergic asthma patients in T-lymphocytes proliferation in vitro. From January to June 2011, 10 subjects with dust mite allergic asthma treated at Third Affiliated Hospital of Soochow University were enrolled. Their peripheral blood monocytes were isolated by Ficoll-Hypaque solution density gradient centrifugation and adherent method. And the adherent monocytes were routinely cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF)+interleukin-4 (IL-4)+tumor necrosis factor-alpha (TNF-α), stimulated with/without interleukin-10 (IL-10), pulsed with dust mite allergen and finally harvested. The cell surface molecules including CD80, CD83, CD86, human leukocyte antigen (HLA)-DR and immunoglobulin-like transcript 2 (ILT2) were detected by immunofluorescent labeling and flow cytometry. And cellular functions were estimated by detecting the capacities of DC uptake antigens with fluorescein isothiocyanate (FITC)-dextran capturing assay. The IL-10 differentiation DC (DC-10) were cultured with autologous peripheral T cells (DC-10 group), either alone (DC-TNF group) or together (combined group) with autologous immunostimulatory DC (DC-TNF). And the impact of this treatment on T-cell responses was assessed for each donor by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di-phenytetrazoliumromide (MTT) assay. The production of interferon-γ (IFN-γ), IL-4, interleukin-5 (IL-5) and interleukin-13 (IL-13) were measured with the quantification of enzyme linked immunosorbent assay (ELISA) kits. In DC-10, the levels of some mature DC's markers (CD80, CD83, CD86 & HLA-DR) decreased, ILT2 increased and there were the higher capacities of up-taking FITC-dextran particle (72.32%±2.93% vs 54.41%±2.95%, P<0.01). Compared with the DC-TNF group (1.74±0.15), the T cell proliferation of the DC-10 group (1.06±0.18) and that of the combined group (1.34±0.16) were significantly inhibited (P<0.01). The secretion levels of IFN-γ, IL-4, IL-5 and IL-10 were (2998±141), (157±17), (2608±254) and (55±11) ng/L in the DC-10 group versus (3223±203), (149±19), (2465±183) and (88±10) ng/L respectively in the combined group. They were all significantly reduced versus (3639±209), (173±16), (2771±183) and (127±11) ng/L in the DC-TNF group (all P<0.01). The IL-10-treated human DC may express a tolerogenic phenotype and induce the allergen tolerance of T cells. And the induction of DC-10 represents a promising target for therapeutic intervention of effectively managing the clinical outcomes of allergic asthma.

Nanoparticle tracking analysis monitors microvesicle and exosome secretion from immune cells

Nanoparticle tracking analysis permits the determination of both the size distribution and relative concentration of microvesicles, including exosomes, in the supernatants of cultured cells and biological fluids. We have studied the release of microvesicles from the human lymphoblastoid T-cell lines Jurkat and CEM. Unstimulated, both cell lines release microvesicles in the size range 70-90 nm, which can be depleted from the supernatant by ultracentrifugation at 100 000 g, and by anti-CD45 magnetic beads, and which by immunoblotting also contain the exosome-associated proteins Alix and Tsg101. Incubation with known potentiators of exosome release, the ionophores monensin and A23187, resulted in a significant increase in microvesicle release that was both time and concentration dependent. Mass spectrometric analysis of proteins isolated from ultracentrifuged supernatants of A23187-treated cells revealed the presence of exosome-associated proteins including heat-shock protein 90, tubulin, elongation factor α1, actin and glyceraldehyde 3-phosphate dehydrogenase. Additionally, treatment of peripheral blood monocyte-derived dendritic cells with bacterial lipopolysaccharide displayed an increase in secreted microvesicles. Consequently, nanoparticle tracking analysis can be effectively applied to monitor microvesicle release from cells of the immune system.

Regulatory T cells form stable and long-lasting cell cluster with myeloid dendritic cells (DC)

Regulatory T cells (Treg) with the capacity to suppress T-cell proliferation exert various effects on T cell function. In addition, Treg have been shown to modulate the phenotype and function of antigen-presenting cells (APC) including dendritic cells (DC), B cells and monocytes/macrophages. However, the specific mechanism(s) of how Treg affect APC have not been entirely identified so far. In this study, we analyzed the interaction of human Treg and effector T cells (Teff) with peripheral blood myeloid and monocyte-derived dendritic cells in vitro. A strong tendency for cell cluster formation between Treg and DC was observed, which was dependent on the adhesion molecules ICAM-1, LFA-3 and ICAM-3. In addition, Treg were found to express higher levels of LFA-1, LFA-2, LFA-3 and ICAM-3 both before and after activation with anti-CD3 antibodies. Using in vitro live cell imaging, we were further able to show that Treg-DC cell clusters, in contrast to Teff-DC clusters, were stable and long lasting. Co-cultures of DC with Treg diminished the up-regulation of activation induced costimulatory molecule expression on DC, and further reduced the production of tumor necrosis factor alpha and stimulated the production of IL-4. In summary, our data indicate that Treg-DC cluster formation might enable Treg to modulate phenotypic and functional characteristics of DC and help to constrain Teff activation.

Pulsing with blast cell lysate or blast-derived total RNA reverses the dendritic cell-mediated cytotoxic activity of cytokine-induced killer cells against allogeneic acute myelogenous leukemia cells.

Immunotherapeutic strategies may be a treatment option in patients with refractory acute myelogenous leukemia (AML) or, in cases of complete remission after conventional therapy regimens, may help to reduce disease recurrence or delay time to progression. Evidence suggests a key role of dendritic cells (DCs) in cancer immunotherapy due to their capacity to present tumour antigens to effector cells. We generated cytokine-induced killer (CIK) cells from healthy donors and examined their responses in vitro in an LDH release assay against three cell lines and allogeneic HLA non-matched blasts from three patients with de novo AML after coincubation with autologous peripheral blood monocyte-derived DCs. Although DCs were unable to enhance CIK cell effects against all three cell lines tested, the cytotoxic activity against the patients’ AML cells increased after coculture with mature DCs, which was significant in two of three patients. However, neither prior pulsing of the DCs with blast cell lysates nor with leukemic cell-derived total RNA further enhanced the lytic capacity of the CIK cells. On the contrary, pulsing reduced or even reversed the cytotoxic activity of the effector cells. This decrease of allogeneic cytotoxicity led us to conclude that monocyte-derived DCs may be useful in autologous or allogeneic vaccine strategies for the treatment of AML or in priming donor lymphocytes in vitro, but unfractionated antigens as pulsing agents may have inhibitory effects on T cell efficiency and their employment in immunotherapeutic strategies for AML seems questionable.

The Novel Neutrophil Differentiation Marker Phosphatidylglucoside Mediates Neutrophil Apoptosis

A new type of glycolipid, phosphatidylglucoside (PtdGlc), was identified as a component of raft-like membrane domains of the human leukemia cell line HL-60. In this study, we show that PtdGlc forms functional domains that are different from those produced by lactosylceramide (LacCer)-enriched lipid rafts. These rafts initiate neutrophil apoptosis. Neutrophils are the only type of human peripheral blood leukocyte or monocyte-derived dendritic cell to express large amounts of PtdGlc on their cell surfaces. PtdGlc was not colocalized with LacCer. Anti-PtdGlc IgM DIM21 did not induce neutrophil chemotaxis or superoxide generation, whereas anti-LacCer IgM T5A7 induced these activities. DIM21, but not T5A7, significantly induced neutrophil apoptosis. DIM21-induced apoptosis was inhibited by specific inhibitors of cysteine-containing aspartate-specific proteases (caspases)-8, -9, and -3 but not by the Src family kinase inhibitor PP1, PIP(3) kinase inhibitor LY294002, NADPH oxidase inhibitor diphenyleneiodonium, superoxide dismutase, or catalase. PtdGlc was colocalized with Fas on the neutrophil plasma membrane. DIM21 and the agonist anti-Fas Ab DX2 induced the formation of large Fas-colocalized clusters of PtdGlc on the plasma membrane. Furthermore, the antagonistic anti-Fas Ab ZB4 significantly inhibited DIM21-induced neutrophil apoptosis. These results suggest that PtdGlc is specifically expressed on neutrophils and mediates apoptosis of these cells, and that the Fas-associated death signal may be involved in PtdGlc-mediated apoptosis.

A Novel In Vitro Method for the Detection and Characterization of Photosensitizers

Photoactivation and binding of photoactive chemicals to proteins is a known prerequisite for the formation of immunogenic photoantigens and the induction of photoallergy. The intensive use of products and the availability of new chemicals, along with an increasing exposure to sun light contribute to the risk of photosensitizing adverse reactions. Dendritic cells (DC) play a pivotal role in the induction of allergic contact dermatitis. Human peripheral blood monocyte derived dendritic cells (PBMDC) were thus perceived as an obvious choice for the development of a novel in vitro photosensitization assay using the modulation of cell surface protein expression in response to photosensitizing agents. In this new protocol, known chemicals with photosensitizing, allergenic or non-allergenic potential were pre-incubated with PBMDCs prior to UVA irradiation (1 J/cm2). Following a 48 h incubation, the expression of the cell surface molecules CD86, HLA-DR and CD83 was measured by flow cytometry. All tested photosensitizers induced a significant and dose-dependent increase of CD86 expression after irradiation compared to non-irradiated controls. Moreover, the phototoxicity of the chemicals could also be determined. In contrast, (i) CD86 expression was not affected by the chosen irradiation conditions, (ii) increased CD86 expression induced by allergens was independent of irradiation and (iii) no PBMDC activation was observed with the non-allergenic control. The assay proposed here for the evaluation of the photoallergenic potential of chemicals includes the assessment of their allergenic, phototoxic and toxic potential in a single and robust test system and is filling a gap in the in vitro photoallergenicity test battery.

Interferon-Alpha Mediates Restriction of Human Immunodeficiency Virus Type-1 Replication in Primary Human Macrophages at an Early Stage of Replication

Type I interferons (IFNα and β) are induced directly in response to viral infection, resulting in an antiviral state for the cell. In vitro studies have shown that IFNα is a potent inhibitor of viral replication; however, its role in HIV-1 infection is incompletely understood. In this study we describe the ability of IFNα to restrict HIV-1 infection in primary human macrophages in contrast to peripheral blood mononuclear cells and monocyte-derived dendritic cells. Inhibition to HIV-1 replication in cells pretreated with IFNα occurred at an early stage in the virus life cycle. Late viral events such as budding and subsequent rounds of infection were not affected by IFNα treatment. Analysis of early and late HIV-1 reverse transcripts and integrated proviral DNA confirmed an early post entry role for IFNα. First strand cDNA synthesis was slightly reduced but late and integrated products were severely depleted, suggesting that initiation or the nucleic acid intermediates of reverse transcription are targeted. The depletion of integrated provirus is disproportionally greater than that of viral cDNA synthesis suggesting the possibility of a least an additional later target. A role for either cellular protein APOBEC3G or tetherin in this IFNα mediated restriction has been excluded. Vpu, previously shown by others to rescue a viral budding restriction by tetherin, could not overcome this IFNα induced effect. Determining both the viral determinants and cellular proteins involved may lead to novel therapeutic approaches. Our results add to the understanding of HIV-1 restriction by IFNα.

Epithelial Cells from Smokers Modify Dendritic Cell Responses in the Context of Influenza Infection

Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza-induced immune response, we established a model using differentiated nasal epithelial cells (NECs) from nonsmokers and smokers, co-cultured with peripheral blood monocyte-derived dendritic cells (mono-DCs) from nonsmokers. NEC/mono-DC co-cultures were infected with influenza A virus and analyzed for influenza-induced immune responses 24 hours after infection. We observed that NECs from smokers, as well as mono-DCs co-cultured with NECs from smokers, exhibited suppressed influenza-induced, interferon-related proteins interferon regulatory factor-7, Toll-like receptor-3, and retinoic acid inducible gene-1, likely because of the suppressed production of IFNα from the NECs of smokers. Furthermore, NEC/mono-DC co-cultures using NECs from smokers exhibited suppressed concentrations of T-cell/natural killer cell chemokine interferon gamma-induced protein 10 (IP-10) after infection with influenza, indicating that NECs from smokers may skew early influenza-induced Th1 responses. In contrast, NEC/mono-DC co-cultures using NEC from smokers contained increased influenza-induced concentrations of the Th2 chemokine thymic stromal lymphopoeitin (TSLP). In addition, NECs from smokers cultured alone had increased influenza-induced concentrations of the Th2 chemokine thymus and activation-regulated chemokine (TARC). Using this model, we demonstrated that in the context of infection with influenza, NECs obtained from smokers create an overall cytokine microenvironment that suppresses the interferon-mediated Th1 response and enhances the TSLP-TARC-mediated Th2 response, with the potential to modify the responses of DCs. Smoking-induced alterations in the Th1/Th2 balance may play a role in developing underlying susceptibilities to respiratory viral infections, and may also promote the likelihood of acquiring Th2 proallergic diseases.

In vitro detection of contact allergens: Development of an optimized protocol using human peripheral blood monocyte-derived dendritic cells

Allergic contact dermatitis is a delayed T-cell mediated allergic response associated with relevant social and economic impacts. Animal experiments (e.g. the local lymph node assay) are still supplying most of the data used to assess the sensitization potential of new chemicals. However, the 7th amendment to the EU Cosmetic Directive will introduce a testing ban for cosmetic ingredients after 2013. In vitro alternative methods are thus being actively developed. Although promising results have been obtained with cell lines, their reduced functionality and inherent genomic instability led us to reinvestigate the use of peripheral blood monocyte-derived dendritic cells (PBMDCs) for the establishment of a reliable in vitro sensitization test. To solve the issues associated with the use of primary cells, the culture and exposure conditions (cytokine concentrations, incubation time, readout, pooled vs. single donors and cytotoxicity) were re-assessed and optimized. Here we propose a stable and reproducible protocol based on PBMDCs. This should allow a wider acceptance of PBMDCs as a reliable test system for the detection of human skin sensitizers and the inclusion of this protocol in an integrated testing strategy.

Direct B-cell Stimulation by Peripheral Blood Monocyte-derived Dendritic Cells in Idiopathic Thrombocytopenic Purpura Patients

Dendritic cells (DCs) play a major role in regulating lymphocytes. The emergence of antiplatelet autoantibodies remains the central pathogenetic mechanism in idiopathic thrombocytopenic purpura (ITP); defective DC functions have been implicated in ITP. The purpose of this study was to assess the contribution of DCs to B-cell hyperactivity in ITP patients. Ten ITP patients were studied before treatment (group untreated) and after treatment (group treated ) with improvement. We compared the effects of monocyte-derived DC from ITP and healthy control (group control) on naive B cells in the presence of lipopolysaccharide, or anti-CD40. We measured the proliferation, antibody production, and expression of activation markers for B cells, as well as DC-secreted B-cell activating factor (BAFF) production. We also measured the serum BAFF level in ITP patients and control. The role of DC-produced BAFF was evaluated with anti-BAFF. Lipopolysaccharide, or anti-CD40, stimulated DCs from group untreated increased B-cell proliferation and antibody production as compared with DCs from group treated and group control. Cell-to-cell contact was not necessary for the augmented effect of the ITP DCs. Anti-CD40 treatment induced a higher production of BAFF in group untreated DCs. Serum BAFF levels, supernatant BAFF from anti-CD40-activated DCs, and BAFF mRNA expression of anti-CD40-activated DCs in group untreated were significantly higher than that in group treated and group control. In ITP patients, there was positive correlation among serum BAFF, supernatant BAFF, and BAFF mRNA levels, and there was negative correlation between serum BAFF, supernatant BAFF, BAFF mRNA levels, and blood platelet count. Blocking BAFF abrogated the effects of ITP DCs on naive B partially. Activated monocyte-derived DCs from ITP patients directly increase B-cell effector functions; this effect depends on soluble factors released by activated DCs and cell-to-cell contact. This might play an important role in the antibody production in ITP. DC-secreted BAFF is involved and might contribute to disease development.

CD40 ligand-triggered human dendritic cells mount interleukin-23 responses that are further enhanced by danger signals

Interleukin (IL)-23 is a heterodimeric cytokine composed of the IL-23-specific subunit p19 and the p40 subunit which also constitutes part of IL-12. IL-23 propagates development of Th17 cells, a novel T cell subset which produces IL-17 but no interferon-γ or IL-4. For both, IL-23 and IL-23-driven IL-17, a crucial role in autoimmune diseases such as experimental autoimmune encephalomyelitis, collagen-induced arthritis, and colitis is well accepted. Recent studies indicate that there is also a role for IL-23 and IL-17 in tumorigenesis, promoting tumor growth and vascularization, and affecting tumor incidence. We show that human CD14+ peripheral blood monocyte-derived dendritic cells (DC), as used for clinical applications in anti-tumor immunization strategies, produce high amounts of IL-23. CD40-triggering of immature and mature DC but not of primary monocytes induced a rapid expression of high levels of IL-23, free p40, and minor levels of IL-12. Upon stimulation of DC subsets with a variety of different danger signals such as single stranded and double stranded RNA, bacterial components or viral infections, IL-23 expression pattern was analyzed. Interestingly, co-stimulation with CD40L enabled IL-23 expression by DC subsets towards danger signals to which they have been unresponsive upon single stimulation. Furthermore, we detected two novel splice variants of the IL-23-specific subunit p19 that could be associated with the regulation of IL-23 expression. Data presented here might have an impact on DC-based cancer vaccination strategies and contribute to a better understanding of the complex regulation of the heterodimeric cytokine IL-23.

The Effect of Gefitinib on Immune Response of Human Peripheral Blood Monocyte-Derived Dendritic Cells

Background: Synergistic antitumor effects of the combined chemoimmunotherapy based on dendritic cells have been reported recently. The aim of this study is to search new applicability of gefitinib into the combination treatment through the confirmation of gefitinib effects on the monocyte derived dendritic cells (moDCs); most potent antigen presenting cell (APC). Methods: Immature and mature monocyte-derived dendritic cell (im, mMoDC)s were generated from peripheral blood monocyte (PBMC) in Opti-MEM culture medium supplemented with IL-4, GM-CSF and cocktail, consisting of TNF-α (10 ng/mL), IL-1β (10 ng/mL), IL-6 (1,000 U/mL) and PGE(2) (1 μ/mL). Various concentrations of gefitinib also added on day 6 to see the influence on immature and mature MoDCs. Immunophenotyping of DCs under the gefitinib was performed by using monoclonal antibodies (CD14, CD80, CD83, CD86, HLA-ABC, HLA-DR). Supernatant IL-12 production and apoptosis of DCs was evaluated. And MLR assay with [3H]-thymidine uptake assay was done. Results: Expression of CD83, MHC I were decreased in mMoDCs and MHC I was decreased in imMoDCs under gefitinib. IL-12 production from mMoDCs was decreased under 10 μM of gefitinib sinificantly. Differences of cell proliferation capacity were not observed in each concentration of geftinib. Conclusion: In spite of decreased expressions of some dendritic cell surface molecules and IL-12 production under 10 μM of gefitinib, significant negative influences of gefitinib in antigen presenting capacity and T cell stimulation were not observed.

Binding immunoglobulin protein‐treated peripheral blood monocyte‐derived dendritic cells are refractory to maturation and induce regulatory T‐cell development

Binding immunoglobulin protein (BiP) has been shown previously to have immunomodulatory functions. Herein we investigated whether BiP could affect the differentiation of monocytes into dendritic cells (DCs) and thence the development of regulatory T cells. Peripheral blood monocyte-derived DCs were matured with lipopolysaccharide in the presence or absence of BiP. DC development and T-cell changes were monitored by flow cytometry and regulatory T-cell function was measured by uptake of tritiated thymidine. More BiP-treated DCs (DC((BiP))s) expressed amounts of intracellular indoleamine 2,3-dioxygenase (IDO) and cell surface leucocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1), retained CD14 expression but down-regulated expression of human leucocyte antigen (HLA)-DR and CD86, and produced copious amounts of interleukin (IL)-10, when compared with control DCs. T cells co-cultured with DC((BiP))s developed regulatory function with increased surface expression of CD4(+) CD25(hi) CD27(hi) but with no concomitant increase in forkhead box P3 (Foxp3). These T cells also showed significantly higher levels of intracellular cytotoxic T-lymphocyte antigen (CTLA)-4. The latter could be inhibited by the presence of the IDO inhibitor 1 methyl tryptophan. The addition of neutralizing anti-IL-10 antibody or the specific mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 reversed the inhibition of DC differentiation by BiP. In conclusion, BiP is an immunomodulator able to arrest inflammation through induction of tolerogenic DCs and subsequent generation of T regulatory cells.

Comparative DNA microarray analysis of human monocyte derived dendritic cells and MUTZ-3 cells exposed to the moderate skin sensitizer cinnamaldehyde

The number of studies involved in the development of in vitro skin sensitization tests has increased since the adoption of the EU 7th amendment to the cosmetics directive proposing to ban animal testing for cosmetic ingredients by 2013. Several studies have recently demonstrated that sensitizers induce a relevant up-regulation of activation markers such as CD86, CD54, IL-8 or IL-1β in human myeloid cell lines (e.g., U937, MUTZ-3, THP-1) or in human peripheral blood monocyte-derived dendritic cells (PBMDCs). The present study aimed at the identification of new dendritic cell activation markers in order to further improve the in vitro evaluation of the sensitizing potential of chemicals. We have compared the gene expression profiles of PBMDCs and the human cell line MUTZ-3 after a 24-h exposure to the moderate sensitizer cinnamaldehyde. A list of 80 genes modulated in both cell types was obtained and a set of candidate marker genes was selected for further analysis. Cells were exposed to selected sensitizers and non-sensitizers for 24 h and gene expression was analyzed by quantitative real-time reverse transcriptase-polymerase chain reaction. Results indicated that PIR, TRIM16 and two Nrf2-regulated genes, CES1 and NQO1, are modulated by most sensitizers. Up-regulation of these genes could also be observed in our recently published DC-activation test with U937 cells. Due to their role in DC activation, these new genes may help to further refine the in vitro approaches for the screening of the sensitizing properties of a chemical.

Reduced ability of hemagglutinin of the CAM-70 measles virus vaccine strain to use receptors CD46 and SLAM

The CAM-70 measles virus (MV) vaccine strain is currently used for vaccination against measles. We examined the fusion-inducing ability of the CAM-70 hemagglutinin (H) protein and found that it was impaired in both CD46- and signaling lymphocyte activation molecule (SLAM)-expressing cells. We also generated recombinant MVs possessing H genes derived from the CAM-70 strain. The CAM-70 H protein impaired viral growth in both CD46- and SLAM-expressing cells. In peripheral blood lymphocytes (PBL) and monocyte-derived dendritic cells (Mo-DC), the CAM-70 strain did not grow efficiently. Infection with recombinant MVs revealed that impaired growth of the CAM-70 strain was attributed to the H gene only partly in PBL and largely in Mo-DC. Thus, impaired fusion-inducing ability of the H protein may be one of the underlying molecular mechanisms resulting in the attenuation of the CAM-70 strain.