Expression Of Dendritic Cells Surface Markers Research Articles

Analysis of the role of palmitoleic acid in acute anterior uveitis

PurposeTo study the role of palmitoleic acid (PA) in the pathogenesis of acute anterior uveitis (AAU). MethodsPA levels in feces from AAU patients were measured by gas chromatography coupled with a mass spectrometer (GC-MS) and compared with samples obtained from healthy individuals. Enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to assess the effect of PA on dendritic cells (DCs) and CD4+T cells obtained from mice, AAU patients and healthy individuals. C57BL/6 mice were fed with PA or vehicle and experimental autoimmune uveitis (EAU) was induced with a human retinal IRBP651-670 peptide. Disease severity of EAU was evaluated by clinical manifestation and histology. Differentiation of splenic Type 1 helper T cells (Th1) and Th17 cells was evaluated by FCM. Tandem mass tag (TMT)-based proteomics analysis was used to identify differentially expressed proteins following incubation of DCs with PA. ResultsThe fecal concentration of PA was increased in AAU patients as compared with healthy individuals. In vitro, PA promoted apoptosis of DCs and inhibited the secretion of TNF-α from mouse bone-marrow-derived dendritic cells (BMDCs) as well as in DCs from AAU patients and healthy individuals. It only decreased DCs surface marker expression and IL-12p70 secretion in BMDCs and healthy individuals DCs but not in AAU patient DCs. PA-treated BMDCs inhibited Th cell differentiation from mouse naïve CD4+T cells and IL-17 and IFN-γ secretion in co-culture supernatants. PA also inhibited the differentiation of Th cells and secretion of IFN-γ and IL-17 in CD4+T cells from mice, AAU patients and healthy individuals. In vivo, PA-treated EAU mice showed milder clinical and histopathological intraocular manifestations as compared with the control group. PA feeding inhibited differentiation of splenic Th17 cells, whereas Th1 cells were not affected. Up to 30 upregulated and 77 downregulated proteins were identified when comparing PA-treated DCs with controls. ConclusionAn increased expression of fecal PA was observed in AAU patients. PA was shown to have immunoregulatory effects on DCs and CD4+T cells and attenuated disease severity in EAU mice.

Effect of mesenchymal stem cell-derived exosomes on the induction of mouse tolerogenic dendritic cells.

Dendritic cells (DCs) orchestrate innate inflammatory responses and adaptive immunity through T‐cell activation via direct cell–cell interactions and/or cytokine production. Tolerogenic DCs (tolDCs) help maintain immunological tolerance through the induction of T‐cell unresponsiveness or apoptosis, and generation of regulatory T cells. Mesenchymal stromal cells (MSCs) are adult multipotent cells located within the stroma of bone marrow (BM), but they can be isolated from virtually all organs. Extracellular vesicles and exosomes are released from inflammatory cells and act as messengers enabling communication between cells. To investigate the effects of MSC‐derived exosomes on the induction of mouse tolDCs, murine adipose‐derived MSCs were isolated from C57BL/6 mice and exosomes isolated by ExoQuick‐TC kits. BM‐derived DCs (BMDCs) were prepared and cocultured with MSCs‐derived exosomes (100 μg/ml) for 72 hr. Mature BMDCs were derived by adding lipopolysaccharide (LPS; 0.1μg/ml) at Day 8 for 24 hr. The study groups were divided into (a) immature DC (iDC, Ctrl), (b) iDC + exosome (Exo), (c) iDC + LPS (LPS), and (d) iDC + exosome + LPS (EXO + LPS). Expression of CD11c, CD83, CD86, CD40, and MHCII on DCs was analyzed at Day 9. DC proliferation was assessed by coculture with carboxyfluorescein succinimidyl ester‐labeled BALB/C‐derived splenocytes p. Interleukin‐6 (IL‐6), IL‐10, and transforming growth factor‐β (TGF‐β) release were measured by enzyme‐linked immunosorbent assay. MSC‐derived exosomes decrease DC surface marker expression in cells treated with LPS, compared with control cells ( ≤ .05). MSC‐derived exosomes decrease IL‐6 release but augment IL‐10 and TGF‐β release (p ≤ .05). Lymphocyte proliferation was decreased (p ≤ .05) in the presence of DCs treated with MSC‐derived exosomes. CMSC‐derived exosomes suppress the maturation of BMDCs, suggesting that they may be important modulators of DC‐induced immune responses.

Exogenous dendritic cells aggravate atherosclerosis via P-selectin/ PSGL-1 pathway

Studies have found that a large number of inflammatory cells, P-selectin, and mature dendritic cells (DCs) are expressed in the damaged and shoulder parts of atherosclerotic plaque, which demonstrates that P-selectin and mature DCs participate in the immune inflammatory response leading to the development of atherosclerosis. However, it is unclear how the above factors interact in this setting. In this study, we investigated the role of P-selectin and its receptor, P-selectin glycoprotein ligand (PSGL)-1 in atherosclerosis, with the finding that DC surface marker expression was consistently high in the P-selectin group while consistently low in the PGSL-1 + DCs group, with CD40 and CD86 expressed by 3.84% and 2.05% for the latter. The highest expression of CD80, CD83, and MHC II was discovered in the DC group, at 7.49%, 3.68%, and 8.98%, respectively. Results of this study are similar to those obtained previously by Ye et al. (2017), which showed larger atherosclerotic lesions in mice that received exogenous DCs, compared with those treated with PBS. In this study, the greatest level of atherosclerosis, fibrosis, and lipid deposition was also seen in mice that received exogenous DCs.

Characteristics of Impaired Dendritic Cell Function in Patients With Hepatitis B Virus Infection.

Dendritic cells (DCs) are antigen-presenting cells with a central role in host immune response. This study analyzed gene expression and DC function in hepatitis B virus (HBV) patients, functions impaired because of HBV, and identified the genes related to these functions. Peripheral blood mononuclear cells from 64 HBV patients and 19 healthy controls were analyzed. Peripheral blood DCs were stained with antibodies against human leukocyte antigen-DR/Lin-1/CD123/CD11c and separated into plasmacytoid DCs (pDCs) and myeloid DCs by fluorescence-activated cell sorting. Using an interferon-gamma enzyme-linked immunospot assay, we analyzed antigen-specific response in HBV-infected patients. Regarding DC function, we analyzed antigen-presenting capacity, cell migration capacity, phagocytic capacity, and cytokine production capacity. DC gene expression was analyzed by microarray to identify genes related to DC function. No difference was found in the number of DCs in peripheral blood between healthy participants and HBV patients. In cell-surface marker analysis, CD80, CD83, CD86, CD40, and C-C motif chemokine receptor 7 expression levels in pDCs were related to the HBV-specific T-cell response. DCs from HBV patients exhibited decreases in antigen-presenting capacity, migration capacity, and cytokine production capacity. In gene expression analysis, immune-related genes with greatly reduced expression levels in chronic hepatitis B patients were identified. Of these genes, interleukin (IL)-6 signal transducer (IL6ST) expression level positively correlated with DC surface marker expression level. Adjustment of IL6ST expression level in DCs and treatment with oncostatin M resulted in recovery of DC function. Conclusion: IL6ST expression was identified as one cause of decline in DC function in HBV patients. Adjustment of IL6 family cytokine signaling may be useful for recovering reduced DC function in HBV infection.

H9N2 avian influenza virus enhances the immune responses of BMDCs by down-regulating miR29c

Avian influenza virus (AIV) of the subtypes H9 and N2 is well recognised and caused outbreaks-due to its high genetic variability and high rate of recombination with other influenza virus subtypes. The pathogenicity of H9N2 AIV depends on the host immune response. Dendritic cells (DCs) are major antigen presenting cells that can significantly inhibit H9N2 AIV replication. MicroRNAs (miRNAs) influence the ability of DCs to present antigens, as well as the ability of AIVs to infect host cells and replicate. Here, we studied the molecular mechanism underlying the miRNA-mediated regulation of immune function of mouse DCs. We first screened for and verified the induction of miRNAs in DCs after H9N2 AIVstimulation. We also constructed miR29c, miR339 and miR222 over-expression vector and showed that only the induction of miR29c lead to a hugely increased expression of surface marker MHCII and CD40. Whilst the inhibition of miR29c, miR339 and miR222 in mouse DCs would repressed the expression of DCs surface markers. Moreover, we found that miR29c stimulation not only up-regulate MHCII and CD40, but also enhance the ability of DCs to activate lymphocytes and secrete cytokines IL-6 or TNF-a. Furthermore, we found that Tarbp1 and Rfx7 were targeted and repressed by miR29c. Finally, we revealed that the inhibition of miR29c marvelously accelerated virus replication. Together, our data shed new light on the roles and mechanisms of miR29c in regulating DC function and suggest new strategies for combating AIVs.

Evaluation of two different dendritic cell preparations with BCG reactivity

Dendritic cells (DCs) play a key-role in the immune response against intracellular bacterial pathogens, including mycobacteria. Monocyte-derived dendritic cells (MoDCs) are considered to behave as inflammatory cell populations. Different immunomagnetic methods (positive and negative) can be used to purify monocytes before their in vitro differentiation and their culture behavior can be expected to be different. In this study we evaluated the reactivity of two dendritic cell populations towards the Bacillus Calmette-Gu?rin (BCG) antigen. Monocytes were obtained from the blood of healthy donors, using positive and negative immunomagnetic separation methods. The expression of DC-SIGN, CD86, CD80, HLA-DR and CD40 on MoDCs was estimated by flow cytometry. The level of IL-12p70, IL-10 and TNF-? was measured by ELISA. Neither of the tested methods affected the surface marker expression of DCs. No significant alteration in immunological response, measured by cytokine production, was noted either. After BCG stimulation, the absence of IL-12, but the IL-23 production was observed in both cell preparations. Positive and negative magnetic separation methods are effective techniques to optimize the preparation of monocytes as the source of MoDCs for potential clinical application.

A possible role for interleukin 37 in the pathogenesis of Behcet's disease.

Interleukin 37 has been found to play a significant regulatory role in the innate immune response. It is not yet known whether IL-37 has also been involved in the development of Behcet's disease (BD), a chronic systemic inflammatory disease. To examine the role of IL-37 in the pathogenesis of BD, a number of experiments were performed. IL-37 expression in peripheral blood mononuclear cells (PBMCs) from BD patients and normal controls was measured by RT-PCR and flow cytometry. Monocyte-derived Dendritic Cells (DCs) were cultured with or without IL-37 and levels of cytokines in the culture supernatants were measured by ELISA. The DC surface markers, reactive oxygen species (ROS) production and mitogen-activated protein kinase (MAPK) activation were measured by flow cytometry. The effect of IL-37-treated DCs on the development of CD4(+) T cells was measured by ELISA and flow cytometry. The results show that both IL-37 mRNA level and protein expression were significantly decreased in PBMCs from active BD patients compared to normal controls. DCs stimulated with rIL-37 showed a decreased expression of IL-6, IL-1β and TNF-α, and a higher production of IL-27. rIL-37 significantly inhibited the production of ROS by DCs and reduced the activation of ERK1/2, JNK and P38 MAPK in DCs. rIL-37-treated DCs remarkably inhibited Th17 and Th1 cell responses as compared to control DCs. rIL-37 did not affect the expression of DC surface markers (CD40, CD86, CD80 and HLA-DR) or IL-10 production by DCs. We conclude that a decreased IL-37 expression in active BD patients may trigger the production of pro-inflammatory cytokines and ROS in association with activation of Th1 and Th17 cells by DCs.

25-Hydroxy vitamin D3 modulates dendritic cell phenotype and function in Crohn’s disease

In Crohn's disease (CrD), vitamin D may help to balance an exaggerated immune response and thereby improve the disease course. The immunomodulating effects depend on the activation of 25-hydroxy vitamin D3 (25-D3), into 1,25-dihydroxy vitamin D3 (1,25-D3). This activation has previously been shown to take place in dendritic cells (DC) from healthy individuals. We hypothesised that DC from CrD patients are able to regulate and control inflammatory responses through 25-D3 activation. During differentiation, monocyte-derived DC from 20 CrD patients were cultured with either 25-D3 or 1,25-D3 and matured with lipopolysaccharide (LPS). We examined DC surface marker expression, cytokine production, and the ability to induce cell proliferation in an allogeneic mixed leukocyte reaction. Following stimulation with LPS, DC exposed to either 25-D3 or 1,25-D3 exhibited lower expression levels of CD80, CD83, CD86, and HLA-DR and diminished TNF-α production compared with DC cultured with LPS alone. In contrast, CD14 expression and IL-6 production were higher following 25-D3 or 1,25-D3 treatment. Compared with LPS alone, both forms of vitamin D3 reduced the ability of DC to activate lymphocytes. Following stimulation with 25-D3, DC from CrD patients displayed a reduced response to LPS with a diminished capability to activate T cells compared with DC stimulated with LPS alone. These data indicate that intrinsic activation of 25-D3 occurs in DC from CrD patients and show that 25-D3 can modulate DC function in CrD. Our data suggest that vitamin D deficiency may contribute to the uncontrolled inflammatory process seen in CrD.

Intravenous anesthetic propofol suppresses prostaglandin E2 production in murine dendritic cells

Propofol is an intravenous anesthetic that is widely used for anesthesia and sedation. Dendritic cells (DC) are one of the crucial immune cells that bridge innate and adaptive immunity, in which DC process antigens during innate immune responses to present them to naïve T-cells, leading to an establishment of adaptive immunity. Prostaglandin (PG)-E2 may be secreted by DC into the microenvironment, considerably influencing DC phenotype and function, and thus determining the fate of adaptive immunity. Since propofol suppresses PGE2 production in murine macrophages, the primary purpose of the present study was to determine whether propofol also suppresses PGE2 production in DC. Assuming a positive finding of such suppression, we tested whether this also leads to alterations of interleukin (IL)-12 and IL-10 production and DC surface marker expression, both of which can be modulated by PGE2. In bone marrow-derived DC, propofol significantly suppressed the PGE2 production after lipopolysaccharide stimulation. Cyclo-oxygenase (COX) protein expression and arachidonic acid release were unaffected, while COX enzyme activity was significantly inhibited by propofol. The propofol-induced COX inhibition did not lead to the increased production of cysteinyl leukotrienes and leukotriene-B4. Endogenous COX inhibition with propofol, as well as with the selective COX-2 inhibitor, NS-398, did not affect IL-12 and IL-10 production from DC. The surface expression of I-Ab and CD40 on DC was not changed, while that of CD86 slightly increased, with both propofol and NS-398; expression of CD80 was not affected with propofol, but increased slightly with NS-398. Finally, endogenous COX inhibition with either propofol or NS-398 did not significantly affect the ability of DC to induce allogeneic T-cell proliferation. It is concluded that the intravenous anesthetic propofol suppresses COX enzyme activity in DC, with no consequences with respect to IL-12/IL-10 production and allogeneic T-cell proliferation, while minimal consequences were observed in surface molecule expression.

Rv0315, a novel immunostimulatory antigen of Mycobacterium tuberculosis, activates dendritic cells and drives Th1 immune responses

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the most deadly infectious diseases, with approximately two million people dying of TB annually. An effective therapeutic method for activating dendritic cells (DCs) and driving Th1 immune responses would improve host defenses and further the development of a TB vaccine. Given the importance of DC maturation in eliciting protective immunity against TB, we investigated whether Rv0315, a newly identified Mtb antigen, can prompt DC maturation. We found that Rv0315 functionally activated DCs by augmenting the expression of the co-stimulatory molecules CD80 and CD86 as well as MHC class I/II molecules. Moreover, it increased DC secretion of the pro-inflammatory cytokines IL-6, IL-1β, and TNF-α. Unlike LPS, however, Rv0315 induced the secretion of IL-12p70, but not IL-10. In addition, Rv0315-treated DCs accelerated the proliferation of CD4(+) and CD8(+) splenic T cells from Mtb-infected mice, with increased levels of IFN-γ, in syngeneic and allogeneic mixed lymphocyte reactions, indicating that Rv0315 contributes to Th1 polarization of the immune response. Importantly, both mitogen-activated protein kinases and nuclear factor κB signaling mediated the expression of DC surface markers and cytokines. Taken together, our results indicate that Rv0315 is a novel DC maturation-inducing antigen that drives T cell immune responses toward Th1 polarization, suggesting that Rv0315 plays a key role in determining the nature of the immune response to TB.

Peripheral blood dendritic cell subtypes are significantly elevated in subjects with asthma

Dendritic cells (DCs) are crucial for the processing of antigens, T lymphocyte priming and the development of asthma and allergy. Smokers with asthma display altered therapeutic behaviour and a reduction in endobronchial DC CD83 expression compared with non-smokers with asthma. No information is available on the impact of smoking on peripheral blood DC profiles. Determine peripheral blood DC profiles in subjects with and without asthma with differing smoking histories. Forty-three asthmatics (17 smokers, nine ex-smokers and 17 never-smokers) and 16 healthy volunteers (nine smokers and seven never-smokers) were recruited. Spirometry, exhaled nitric oxide and venesection was performed. DC elution was by flow cytometry via the expression of DC surface markers [plasmacytoid (pDC) (BDCA-2, CD303), type 1 conventional (cDC) (BDCA-1, CD1c), and type 2 cDC (BDCA-3, CD141)]. Subjects with asthma displayed increases in all DC subtypes compared with normal never-smokers: [type 1 cDCs - asthma [median% (IQR)]: 0.59% (0.41, 0.74), normal never-smokers: 0.35% (0.26, 0.43), P=0.013]; type 2 cDCs - asthma: 0.04% (0.02, 0.06), normal never-smokers: 0.02% (0.01, 0.03), P=0.008 and pDCs - asthma: 0.32% (0.27, 0.46), normal never-smokers: 0.22% (0.17, 0.31), P=0.043, and increased pDC and type 1 cDCs compared with normal smokers. Smoking did not affect DC proportions in asthma. Cigarette smoking reduced pDC proportions in normal subjects [normal never-smokers: 0.22% (0.17, 0.31); normal smokers: 0.09% (0.08, 0.15), P=0.003]. This study shows for the first time that subjects with asthma display a large increase in peripheral blood DC proportions. Cigarette smoking in asthma did not affect the peripheral blood DC profile but did suppress pDC proportions in non-asthmatic subjects. Asthma is associated with a significant increase in circulating DCs, reflecting increased endobronchial levels and the importance of DCs to the development and maintenance of asthma. (Clinical trials.gov identifier: NCT00411320)

Atrial Natriuretic Peptide Receptor Signaling Plays a Critical Role in Induction of Tolerogenic Dendritic Cells

RATIONALE: Atrial natriuretic peptide (ANP) is an important endogenous hormone, controlling inflammation and immunity by acting through dendritic cells (DCs), however, the mechanisms remain unclear. We sought to determine whether ANP receptor, NPRA, signaling modulates DC function and analyze the downstream signaling events. METHODS: The role of NPRA signaling in modulating DC function was examined using NPRA-signaling inhibiting peptide NP73-102. Immunoblotting and immunoprecipitation assays of human monocyte-derived DC (hmDC) were used to define the potential downstream molecules and the protein interactions among these molecules involved in this signaling pathway. Regulatory T cell phenotype and function were analyzed using flow cytometry and BrdU proliferation ELISA assay. Adoptive transfer studies in mice involved cytokine assays and immunohistochemical analyses. RESULTS: Attenuation of NPRA signaling in DC generates more Treg in our in vitro T-DC co-culture system. Analysis of DC surface marker expression and in vitro phagocytosis suggested that DC maturation was unaffected significantly by modulated NPRA signaling. NP73-102-mediated increases in levels of IL-10 and TGF-β, but not of IL-12 and IFN-γ, compared to those in cells given ANP or vector alone. DC adoptive transfer studies in mice indicated that NPRA signaling in DCs primes DCs to induce Treg cells and attenuated lung inflammation. NPRA signaling is associated with TLR-2, SOCS3 and STAT3 and affects induction of IL-6, IL-10 and TGF-β but not IL-12. CONCLUSIONS: TLR2 and SOCS3 are integral to NPRA signaling in DCs leading to immunity or tolerance induction.

인간 골수성 백혈병 세포에서 Flt-3 수용체 리간드에 의한 CD11c 발현의 증가

CD11c and costimulatory molecules such as CD80 and CD86 express mainly in dendritic cells (DCs). In this study, we investigated the biologic effects of recombinant Fms-like tyrosine kinase-3 (Flt-3) ligand on the expression of DC surface markers, including CD11c in leukemia cell lines, such as KG-1, HL-60, NB4, and THP-1 cells. The expression of the Flt-3 receptor was found in NB4 and HL-60 cells, as well as KG-1 cells, but not in THP-1 cells. When KG-1 cells were cultured in a medium containing Flt-3 ligand or granulocyte macrophage-colony stimulating factor (GM-CSF) plus tumor necrosis factor (TNF)-α, cell proliferation was inhibited and the expression levels of CD11c, major histocompatibility complex (MHC)-Ⅰ, and MHC-Ⅱ were increased in the cells. Flt-3 ligand also increased the expression level of CD11c on HL-60 and NB4 cells, but not on THP-1 cells. In comparison with CD11c expression, the expression level of CD11b on KG-1 cells, but not on NB4 and HL-60 cells, was slightly increased by Flt-3 ligand. Flt-3 ligand induced phosphorylation of extracellular signal-regulated kinase-1/2 (ERK-1/2) and p38-mitogen-activated protein kinase (p38-MAPK) in KG-1 cells, and the up-regulation of CD11c expression by Flt-3 ligand in the cells was abrogated by PD98059, an inhibitor of MEK. The results suggest that Flt-3 ligand up-regulates DC surface markers on CD34? myelomonocytic KG-1 cells, as well as promyelocytic leukemia cells, and that the differentiation of the leukemia cells into DC-like cells by Flt-3 ligand is mediated by ERK-1/2 activity.

Cryopreservation of Monocytes Is Superior to Cryopreservation of Immature or Semi-mature Dendritic Cells for Dendritic Cell-based Immunotherapy

Cryopreservation of immature or mature dendritic cells (DC) has been proposed as a suitable method to gain large numbers of DC for immunotherapeutic trials against cancer. However, clinical studies using cryopreserved DC have demonstrated only limited success so far. The aim of this study was to investigate whether cryopreservation of monocytes elicits more potent DC and whether these DC are comparable to freshly generated DC preparations. Monocytes, either separated immunomagnetically or by means of leukapheresis and elutriation, were differentiated into DC and cryopreserved at various developmental stages. DC preparations were analyzed regarding recovery, viability, phenotype, and functional properties. In contrast to DC frozen at their immature or semi-mature state, generation of DC from cryopreserved monocytes elicited viability values comparable with freshly generated DC. Furthermore, using frozen monocytes for DC differentiation revealed improved expression of DC surface markers and interleukin-12p70 secretion as compared with DC generated from frozen immature or frozen semi-mature DC. Impaired phenotypical appearance of the latter DC variants was further substantiated by functional analysis. T-cells cocultured with these DC showed decreased expression of interferon-gamma and granzyme B, and lowered proliferation when compared with T-cells cocultured with DC generated from frozen monocytes or DC generated from freshly isolated monocytes. Induction of regulatory T-cell populations was negligible among all investigated DC preparations. These findings may further improve DC-based immunotherapeutical protocols. Cryopreservation of unchallenged monocytes enables targeted therapy by loading DC with varying antigenic compositions in case of tumor escape during treatment.

Ovarian Cancer-derived Glycodelin Impairs In Vitro Dendritic Cell Maturation

Local immunosuppressive mechanisms shape the tumor microenvironment and contribute to carcinogenesis. In ovarian cancer such mechanisms have been shown to influence survival. Dendritic cells (DCs) are central immunity regulators and induce potent cytotoxic T-cell responses as well as peripheral tolerance depending on modulatory stimuli. Here, we show that ovarian cancer-derived glycodelin (Gd), a glycoprotein that physiologically modulates local immunity in early pregnancy, induces a tolerogenic DC phenotype. Gd was isolated with high performance liquid chromatography from the malignant ascites of ovarian cancer patients. DCs were generated from monocytes of healthy donors and exposed to Gd with or without an inflammatory stimulus (tumor necrosis factor-alpha and interleukin 1-beta). We investigated the effect of Gd on DC surface marker expression, endopinocytotic activity, cytokine profile, and lymphoproliferative activity. DCs that were exposed to Gd altered their phenotype as seen by a differential expression of costimulatory molecules, whereas expression of DC-specific intercellular adhesion molecule 3-grabbing nonintegrin, a marker of an immature phenotype, was increased. Functional data provided further evidence for the immature/tolerogenic properties of Gd-pretreated DCs. Antigen uptake was retained, production of interleukin-10 was increased, and lymphoproliferative activity was reduced. This effect was reversible by adding Gd-blocking antibodies. Gd, which is found in the malignant ascites of ovarian cancer patients, induces a tolerogenic phenotype in DC, thereby shaping an immunodeficient tumor micromilieu.

Maturation of dendritic cells induced by nano-liposomes containing imiquimod

Background: Imiquimod (IMQ), a small-molecule immune response modifier of the imidazoquinoline family, has been shown to activate functional maturation of dendritic cells (DCs) through the Toll-like receptor (TLR)7- signaling pathway. IMQ has the potential to develop into a novel vaccine adjuvant. Currently, a topical cream of IMQ has been approved for the treatment of genital warts, superficial basal cell carcinoma and actinic keratosis. Objective: To develop nano-liposomes with encapsulated IMQ as a new vaccine adjuvant. Methods: Two types of nano-lipid particles containing IMQ which differed in their surface charge (i.e. positively and negatively charged liposomes) were prepared and characterized. Murine bone marrow derived DCs were cultured and treated with liposomal formulations. The surface marker expression of DCs was then determined by flow cytometry. Results: Both liposomes with IMQ encapsulated enhanced DCs maturation as determined by the surface marker expression of DCs (CD80, CD86, and MHC class II). Cationic liposomes induced high levels of surface marker expression of DCs in similar levels as those induced by lipopolysaccharides (LPS). Conclusion: The novel IMQ formulation, nano-liposome, has the benefits of simplicity and it enhances the adjuvant effect of IMQ.

Impaired Maturation of Monocyte‐derived Dendritic Cells from Birch Allergic Individuals in Association with Birch‐specific Immune Responses

Optimal activation of T lymphocytes requires a costimulatory signal provided by the interaction of molecules on the surface of T cells with their ligands expressed on dendritic cells (DC). We investigated whether DC differentiated from monocytes from healthy and birch allergic asthmatic individuals and further maturated by stimulation with cat and birch allergens and LPS differ in their phenotypic receptor expression. Similar expression of DC surface markers, including HLA-DR, CD80, CD86, CD83, CD1a and CD11c, was detected in monocyte-derived DC from allergic and healthy individuals. Cells from healthy donors stimulated either antigen showed a similar activation of the CD80 and double CD80/CD86 costimulatory molecules when compared with non-stimulated cells. In the case of cells from allergic individuals, birch allergen was unable to produce the same increased expression of CD80 alone or in combination with CD80/CD86, in comparison with cells stimulated with cat and LPS. Levels of IL-6, IL-8, IL-10, MCP-1/MCAF and MIP-1beta were similar in the supernatant of non-stimulated DC from both groups of subjects. By contrast, the spontaneous secretion of IL-12p70 and TNF-alpha was higher in the supernatant of DC from healthy subjects when compared with that from allergic individuals. Stimulation with birch and LPS resulted in an increased secretion of IL-12p70 in samples from healthy when compared with that in allergic individuals. The results suggest an impaired specific maturation of DC from birch allergic individuals in association with birch-specific immune responses. Lower secretion of IL-12p70 from birch-stimulated DC from allergic individuals suggests that not only maturation, but also the specific Th1 function of these cells seems to be affected in those individuals.

Immunosuppressive effect of pregnant mouse serum on allostimulatory activity of dendritic cells

In normal pregnancy, the maternal immune system is directed towards tolerance or suppression in order to prevent rejection of the semi-allogenic fetus. Antigen-presenting cells, especially dendritic cells (DCs), are key cells in initiation and regulation of immune responses. The presence of potent immunostimulatory DCs in the decidual tissue of pregnancy has been demonstrated. The aim of this study was to determine how allostimulatory activity of DCs could be affected during pregnancy. DCs were isolated from spleen of pregnant or non-pregnant Balb/c mice and co-cultured with allogenic T lymphocytes prepared from brachial lymph nodes of C57BL/6 mice. Some cultures of non-pregnant female DCs were treated by 2.5% serum obtained from pregnant mice at early, middle or late gestational periods, and were used in the same mixed lymphocyte reaction (MLR) settings. Cell proliferation was measured by 3H-thymidine incorporation, and cytokine production measured in supernatants of MLR cultures using ELISA. The effect of pregnant mouse serum on expression of DC surface markers was evaluated by flow cytometry. No significant difference was found between stimulatory potential of splenic DCs from pregnant and non-pregnant mice in induction of allogenic T cell proliferative response. Moreover, serum of early or late pregnancy did not have any effect on DC function in comparison with non-pregnant mouse serum, while mid-pregnancy serum significantly inhibited allostimulatory activity of DCs. IFNγ production in co-culture of DCs treated with pregnant mouse serum was significantly lower than that of the control group; however, no significant difference in IL-10 production was observed. Treatment of DCs with pregnant mouse serum did not influence the percentage of cells expressing MHC-II, CD86, CD8α or CD11b. However, a marked reduction of the mean fluorescence intensity of MHC-II was observed. Collectively, our results concerning the diminished capacity of DCs to induce production of Th1 cytokines and allogenic T cell proliferation after treatment with pregnant mouse serum reveal a new way of immunologic tolerance against the semi-allogenic fetus.

Immunomodulatory Effects ofCallophyllis japonicaEthanol Extract on Dendritic Cells

A red seaweed, Callophyllis japonica has been traditionally eaten in the oriental area. In a recent study, it has been demonstrated that the ethanol extract of C. japonica have antioxidant activity. However, there are few studies about the effects of C. japonica on the function of immune cells. We investigated the immunomodulatory effects of C. japonica on the function of dendritic cells, the potent antigen-presenting cells. Methods: Bone marrow-derived dendritic cells (DCs) were used and the viability was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and trypan blue exclusion test. Cytokine and nitric oxide (NO) levels were determined by using ELISA and Griess reagent, respectively. The expression levels of DC surface markers were measured by flow cytometric analysis. Results: C. japonica ethanol extract did not significantly affect the DCs viability and the IL-12 production from DCs, irrespective of the presence of lipopolysaccharide (LPS). In addition, it did not significantly change the expression of DC surface markers. However, C. japonica ethanol extract significantly inhibited the LPS-induced NO production and also increased the proliferation of allogeneic lymphocytes activated by DCs. Conclusion: Our data suggests that C. japonica ethanol extract enhances the proliferation of allogeneic lymphocytes activated by DCs which is associated with inhibition of NO production from DCs induced by LPS.

Comparison of allergen-stimulated dendritic cells from atopic and nonatopic donors dissecting their effect on autologous naive and memory T helper cells of such donors

Background: Because of their production of IL-12, mature dendritic cells (DC) are potent inducers of TH1 responses. However, recent reports have demonstrated that DCs can also induce TH2 differentiation. Objective: In the current study we investigated which immune response is induced by DCs in naive CD45RA+ or memory CD45R0+ CD4+ T cells from atopic individuals (patients with grass pollen, birch pollen, or house dust mite allergy) compared with nonatopic control subjects. Methods: Immature DCs, generated from peripheral blood monocytes from atopic and nonatopic donors, were pulsed with the respective allergen and fully matured. Then the mature DCs were cocultured in vitro with autologous naive (CD45RA+) and memory (CD45R0+) CD4+ T cells and cytokine and IgE production were measured by ELISA. Results: After the second restimulation with allergen-pulsed DCs, naive as well as memory autologous CD4+ T cells from atopic but not from nonatopic donors showed an enhanced production of the TH2-type cytokines IL-4, IL-5, and IL-10, resulting in an increased IgE production, whereas IFN-γ production and proliferation were not different. IL-12 production and surface marker expression of DCs derived from atopic and nonatopic donors did not differ and addition of neutralizing anti-IL-12 mAbs did not increase IL-4 but diminished IFN-γ production. Conclusion: These data indicate that mature DCs are able to induce naive and activate allergen-specific T helper cells to produce TH2 cytokines if the T cells are derived from atopic donors. This phenomenon is not due to diminished IL-12 production by DCs of atopic donors. (J Allergy Clin Immunol 2000;105:988-96.)