Analysis of the interactions between the peptides from secreted human CKLF1 and heparin using capillary zone electrophoresis.

The Chemokine-like factor 1 (CKLF1) is a novel human cytokine and exhibits chemotactic activities on leukocytes. Two peptides named CKLF1-C27 and CKLF1-C19, were obtained from secreted CKLF1. In this study, a selective high-performance analytical method based on capillary zone electrophoresis (CZE) to investigate interactions between heparin and CKLF1-C27/CKLF1-C19 was developed. Samples containing CKLF1-C27/CKLF1-C19 and heparin at various ratios were incubated at room temperature and then separated by CZE with Tris-acetate buffer at pH 7.2. Both qualitative and quantitative characterizations of the binding were determined. The binding constants of the interactions between CKLF1-C27/CKLF1-C19 and heparin were calculated as (3.38 +/- 0.49) x 10(5) M(-1) and (1.10 +/- 0.02) x 10(5) M(-1) by Scatchard analysis. To study structural requirements, CKLF1-C19pm and CKLF1-C19km have been synthesized, and their interactions with heparin have been studied by CZE. We found that the Pro or Lys to Ala substitution within the residues of CKLF1-C19 (CKLF1-C19pm or CKLF1-C19km) strongly decreased or abolished its interaction with heparin, suggesting that the residues of Pro affect the affinity of CKLF1-C19 for heparin, and the residues of Lys of CKLF1-C19 play the important role for the interaction of CKLF1-C19 and heparin, respectively. The methodology presented should be generally applicable to study peptides and heparin interactions quantitatively and qualitatively. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Dendritic cells derived from peripheral monocytes express endothelial markers and in the presence of angiogenic growth factors differentiate into endothelial-like cells

CD14-positive monocytes obtained from human peripheral blood were cultured with GM-CSF and IL-4. During the early culture phase immature dendritic cells (DCs) developed which not only expressed CD1a, HLA-DR and CD86, but also expressed the endothelial cell markers von Willebrand factor (vWF), VE-cadherin and VEGF receptors Flt-1 and Flt-4. Further maturation of DCs was achieved by prolonged cultivation with TNFalpha. These cells showed typical DC morphology and like professional antigen-presenting cells (APCs) expressed CD83 and high levels of HLA-DR and CD86. However, if immature DCs were grown with VEGF, bFGF and IGF-1 on fibronectin/vitronectin-coated culture dishes, a marked change in morphology into caudated or oval cells occurred. In the presence of these angiogenic growth factors the cultured cells developed into endothelial-like cells (ELCs), characterized by increased expression of vWF, KDR and Flt-4 and a disappearance of CD1a and CD83. Addition of IL-4 and Oncostatin M also increased VE-cadherin expression, and the loosely adherent cells formed clusters, cobblestones and network-like structures. vWF- expressing ELCs mainly originated from CD1a-positive cells, and VEGF was responsible for the decrease in the expression of the DC markers CD1a and CD83. In mixed leukocyte cultures, mature DCs were more potent APCs than ELCs. Moreover, Ac-LDL uptake, and the formation of tubular structures on a plasma matrix was restricted to ELCs. These results suggest that in the presence of specific cytokines immature DCs have the potential to differentiate along different lineages, i.e. into a cell type resembling ELCs.     

The cellular pathway of CD1e in immature and maturing dendritic cells

Dendritic cells (DCs) present antigens to T cells via CD1, HLA class I or class II molecules. During maturation, HLA class II-restricted presentation is optimized. The relocalization of CD1e from Golgi to endosomal compartments during DC maturation suggests also an optimization of the antigen-presentation pathway via CD1 molecules. We here detail the biosynthesis and cellular pathway of CD1e in immature and maturing DCs. Unlike the other CD1 molecules, CD1e was found to reach late endosomes through sorting endosomes, without passing through the plasma membrane in either immature or maturing cells. After induction of DC maturation, CD1e disappeared rapidly from the Golgi and was transiently localized in HLA-DR+ vesicles, while the number of CD1e+/CD1b+ compartments increased for at least 20 h. High-resolution light microscopy showed that, in immature DCs, CD1e+ vesicles were often in close apposition to EEA1+ or HLA-DR+ compartments, while CD1e displayed a nearly exclusive distribution in the lysosomes of mature DCs, a finding corroborated by immunoelectron microscopy. During maturation, CD1e synthesis progressively declined, while the endosomal cleavage of CD1e still occurred. Thus, CD1e displays peculiar properties, suggesting an unexpected role among the family of CD1 antigen-presenting molecules.     

MicroRNA-146a and MicroRNA-146b Regulate Human Dendritic Cell Apoptosis and Cytokine Production by Targeting TRAF6 and IRAK1 Proteins

We have previously reported 27 differentially expressed microRNAs (miRNAs) during human monocyte differentiation into immature dendritic cells (imDCs) and mature DCs (mDCs). However, their roles in DC differentiation and function remain largely elusive. Here, we report that microRNA (miR)-146a and miR-146b modulate DC apoptosis and cytokine production. Expression of miR-146a and miR-146b was significantly increased upon monocyte differentiation into imDCs and mDCs. Silencing of miR-146a and/or miR-146b in imDCs and mDCs significantly prevented DC apoptosis, whereas overexpressing miR-146a and/or miR-146b increased DC apoptosis. miR-146a and miR-146b expression in imDCs and mDCs was inversely correlated with TRAF6 and IRAK1 expression. Furthermore, siRNA silencing of TRAF6 and/or IRAK1 in imDCs and mDCs enhanced DC apoptosis. By contrast, lentivirus overexpression of TRAF6 and/or IRAK1 promoted DC survival. Moreover, silencing of miR-146a and miR-146b expression had little effect on DC maturation but enhanced IL-12p70, IL-6, and TNF-α production as well as IFN-γ production by IL-12p70-mediated activation of natural killer cells, whereas miR-146a and miR-146b overexpression in mDCs reduced cytokine production. Silencing of miR-146a and miR-146b in DCs also down-regulated NF-κB inhibitor IκBα and increased Bcl-2 expression. Our results identify a new negative feedback mechanism involving the miR-146a/b-TRAF6/IRAK1-NF-κB axis in promoting DC apoptosis.     

Donor BMSC‐derived small extracellular vesicles relieve acute rejection post‐renal allograft through transmitting Loc108349490 to dendritic cells

Bone marrow‐derived mesenchymal stem cell (BMSC)‐derived small extracellular vesicles (sEVs) are potent candidates for the suppression of acute rejection post‐renal allograft and have been reported to halt dendritic cells (DCs) maturation. However, whether BMSC‐derived sEVs mitigate acute rejection post‐renal allograft by targeting DCs is still unclear. In this study, donor BMSC‐derived sEVs (sEVs) relieved the inflammatory response and suppressed mature DCs (mDCs) location in kidney grafts, and increased regulatory T (Treg) cell population in the spleens of the rats that underwent kidney allograft. In lipopolysaccharide (LPS)‐stimulated immature DCs (imDCs), sEVs suppressed the maturation and migration of DCs and inactivated toll‐like receptor 4 (TLR4) signaling. Compared with LPS‐treated imDCs, imDCs treated with LPS+sEVs promoted CD4⁺T cells differentiated toward Treg cells. Subsequently, we found that Loc108349490, a long non‐coding RNA (lncRNA) abundant in sEVs, mediated the inhibitory effect of sEVs on DC maturation and migration by promoting TLR4 ubiquitination. In rats that underwent an allograft, Loc108349490 deficiency weakened the therapeutic effect of sEVs on acute rejection. The present study firstly found that sEVs alleviated acute rejection post‐renal allograft by transferring lncRNA to DCs and screened out the functional lncRNA loaded in sEVs was Loc108349490.     

骨髓间充质干细胞通过JAK／STAT3信号通路抑制树突状细胞成熟

骨髓间充质干细胞（bone marrow mesenchymal stem cells,bMSCs）具有自我更新、支持造血、多向分化和低免疫原性等特点,在调控树突状细胞（dendritic cells,DCs）成熟的过程中发挥重要作用。为了探讨bMSCs调控DCs成熟的机制,本研究通过分离培养正常捐献者bMSCs,并分离获取外周静脉血单个核细胞,诱导未成熟的树突状细胞（immature dendritic cells,imDCs）和成熟的树突状细胞（mature dendritic cells,mDCs）生成。根据Genebank中人STAT3全长基因序列,设计针对STAT3的siRNA。根据培养条件不同设计实验分组：正常bMSCs与imDCs共培养（阴性对照组）,转染siRNA的bMSCs与imDCs共培养（siRNA组）、加入JAK/STAT通路抑制剂AG490的bMSCs与imDCs共培养（AG490组）、加入TNF-α诱导的mDCs（阳性对照组）共4组,共培养72 h,流式细胞术分析DCs表型变化,ELISA检测培养液上清中IL-12水平变化。结果显示,阴性对照组不表达CD40、CD80、CD83、CD86和HLA DR标志树突细胞成熟的分子,而表达CD11b,其表型与imDCs一致;而siRNA组和AG490组的DCs表达CD40、CD80、CD83、CD86和HLA-DR等标志分子,而不表达CD11b,其表型与TNF-α诱导成熟的mDCs表型一致;siRNA组、AG490组和阳性对照组的IL-12水平较阴性对照组的IL-12水平显著升高（P＜0.05）,但siRNA组、AG490组和阳性对照组之间无明显差异（P＞0.05）。以上结果表明,通过siRNA和抑制剂AG490阻断bMSCs中JAK/STAT3通路促进了imDCs的成熟,提示bMSCs通过JAK/STAT3通路参与调控imDCs成熟。     

CD1 molecules efficiently present antigen in immature dendritic cells and traffic independently of MHC class II during dendritic cell maturation

Upon exposure to Ag and inflammatory stimuli, dendritic cells (DCs) undergo a series of dynamic cellular events, referred to as DC maturation, that involve facilitated peptide Ag loading onto MHC class II molecules and their subsequent transport to the cell surface. Besides MHC molecules, human DCs prominently express molecules of the CD1 family (CD1a, -b, -c, and -d) and mediate CD1-dependent presentation of lipid and glycolipid Ags to T cells, but the impact of DC maturation upon CD1 trafficking and Ag presentation is unknown. Using monocyte-derived immature DCs and those stimulated with TNF-alpha for maturation, we observed that none of the CD1 isoforms underwent changes in intracellular trafficking that mimicked MHC class II molecules during DC maturation. In contrast to the striking increase in surface expression of MHC class II on mature DCs, the surface expression of CD1 molecules was either increased only slightly (for CD1b and CD1c) or decreased (for CD1a). In addition, unlike MHC class II, DC maturation-associated transport from lysosomes to the plasma membrane was not readily detected for CD1b despite the fact that both molecules were prominently expressed in the same MIIC lysosomal compartments before maturation. Consistent with this, DCs efficiently presented CD1b-restricted lipid Ags to specific T cells similarly in immature and mature DCs. Thus, DC maturation-independent pathways for lipid Ag presentation by CD1 may play a crucial role in host defense, even before DCs are able to induce maximum activation of peptide Ag-specific T cells.     

Intermediate maturation of Mycobacterium tuberculosis LAM-activated human dendritic cells

Contrasting observations raise the question of the role of mycobacterial derived products as compared with the whole bacterium Mycobacterium tuberculosis on maturation and function of human dendritic cells (DCs). DC-SIGN has been identified as the key DC receptor for M. tuberculosis through its interaction with the mannosylated lipoarabinomannan (ManLAM). Although ManLAM is a major mycobacterial component released from infected antigen-presenting cells, there is no formal evidence yet for an effect of ManLAM per se on DC maturation and function. DCs activated with purified ManLAM displayed an intermediate maturation phenotype as compared with lipopolysaccharide fully matured DCs with reduced expression of MHC class I and class II molecules, CD83 and CD86 and of the chemokine receptor CCR7. They were sensitive to autologous natural killer (NK) lysis, thus behaving like immature DCs. However, ManLAM-activated DCs lost phagocytic activity and triggered priming of naive T-cells, confirming their intermediate maturation. Partial maturation of ManLAM-activated DCs was overcome by triggering the CD40/CD40L pathway as a second signal, which completed maturation phenotypically and abolished autologous NK lysis susceptibility. Altogether, these data provide evidence that ManLAM may induce a partial maturation phenotype on non-infected bystander DCs during infection suggesting that ManLAM released from infected cells might impair adaptive immune response towards M. tuberculosis.     

Dexamethasone selectively inhibits differentiation of cord blood stem cell derived-dendritic cell (DC) precursors into immature DCs

Perinatal dexamethasone (Dx) alters the immune system leading to increased infections and developmental abnormalities. Dendritic cells (DCs) derived from cord-blood monocytes are especially Dx sensitive and we sought to determine the effects of Dx on cord-blood CD34+-DCs. Distinct stages of cord-blood CD34+-DC development were delineated: pre-DC, immature, and mature DCs. Dx added during development of pre-DCs did not suppress precursor number, or translocate the glucocorticoid receptor (GcR) from the cytoplasm to the nucleus. However, Dx added during pre-DCs differentiation into immature DCs, prompted GcR translocation to the nucleus, enhanced DC apoptosis, suppressed differentiation to CD1a+ cells, inhibited expression of CD86, reduced subsequent CD83 expression, maintained DC endocytic activity, suppressed IL-6 secretion, enhanced IL-10 secretion, and reduced DC-mediated T cell stimulation. Dx added during the maturation stage caused less dramatic effects. Thus, Dx stalled maturation, selectively induced apoptosis of developing DCs and the sensitivity peaked during pre-DCs differentiation into immature DCs.     

Oncostatin M induces dendritic cell maturation and Th1 polarization

Oncostatin M (OSM) is a pleiotropic cytokine and a member of the gp130/IL-6 cytokine family that has been found to be involved in both pro- and anti-inflammatory responses in cell-mediated immunity. Maturation of dendritic cells (DCs) is crucial for initiation of primary immune responses and is regulated by several stimuli. In this study, the role of OSM in the phenotypic and functional maturation of DCs was evaluated in vitro. Stimulation with OSM upregulated the expression of CD80, CD86, MHC class I and MHC class II and reduced the endocytic capacity of immature DCs. Moreover, OSM induced the allogeneic immunostimulatory capacity of DCs by stimulating the production of the Th1-promoting cytokine IL-12. OSM also increased the production of IFN-γ by T cells in mixed-lymphocyte reactions, which would be expected to contribute to the Th1 polarization of the immune response. The expression of surface markers and cytokine production in DCs was mediated by both the MAPK and NF-κB pathways. Taken together, these results indicate that OSM may play a role in innate immunity and in acquired immunity by enhancing DCs maturation and promoting Th1 immune responses.     

Analysis of the in vivo dendritic cell response to the bacterial superantigen staphylococcal enterotoxin B in the mouse spleen

To investigate the in vivo effects of Staphylococcal enterotoxin B (SEB) on dendritic cells (DCs) in the spleen, a single dose of SEB (50 microg/kg) was administered to BALB/c mice by intraperitoneal injection. Afterwards, the mice were sacrificed at 2, 6 and 24 hr, 2, 4, 7 and 15 days, and the spleens were removed. The immunocytochemical characterization of the cells was carried out using various monoclonal antibodies in cryostat-cut sections. The distribution patterns of DCs and their major costimulatory molecules, CD80, CD86 and CD40 in the spleen were identified, and the evidence for maturation of DCs in vivo in response to SEB was obtained. It was found that systemic administration of SEB induced the migration of most of the immature, splenic DCs from the marginal zone to the periarterial lymphatic sheath within 6 hr. This movement paralleled a maturation process, as assessed by upregulation of CD40, CD80 and CD86 expression in the interdigitating dendritic cells (IDCs). The upregulation of costimulatory molecule expression was conspicuous only in DCs in contrast to other antigen-presenting cells (APCs) such as macrophages and B cells which did not show any significant alterations in their costimulatory molecule expression. We also demonstrated the temporal expression pattern of these costimulatory molecules on the activated DCs. The upregulation of costimulatory molecules on DCs reached a peak level 6 hr after SEB injection, while the increase in number of T cells expressing T cell receptor V138 reached a peak level on day 2 after SEB treatment. In conclusion, we demonstrated the in vivo DC response to SEB in the mouse spleen, especially a potent stimulative effect of SEB on DCs in vivo, a temporal distribution pattern of DCs as well as T cells including TCR Vbeta8+ T cells, and a differential expression pattern of costimulatory molecules on the activated DCs. The results of the present study indicate that DCs are the principal type of APCs which mediate T cell activation by SAg in vivo, and that each costimulatory molecule may have different role in the activation of DCs by SAg. Thus, it is plausible to speculate that DCs play a critical role in the T cell clonal expansion by SAgs and other SAg-induced immune responses in vivo.     

Endocytosis of micro- and nanosized particles in vitro by human dendritic cells

The ability of human dendritic cells (DC) to uptake synthetic micro- and nanosized particles was assessed by flow cytometry and fluorescent microscopy. DCs were differentiated in vitro from blood monocytes in the presence of recombinant cytokines. Further maturation of DC in culture after the addition of maturation factors resulted in the increased expression of HLA-DR and co-stimulatory molecules CD80, CD83, CD86, in comparison with immature DC. Active internalization of Fluoresbrite-YG fluorescent microbeads (0.2 μm) was noted for immature but not mature DCs. The decrease of endocytic activity after DC maturation correlated with the reduced expression of CD209, the surface membrane receptor participating in phagocytosis. Unlike microparticles, the uptake of nanoscale Quantum dots-655 did not depend on the stage of DC maturation and probably was mediated by a different endocytosis mechanism.     

Chemokine-Like Factor 1-Derived C-Terminal Peptides Induce the Proliferation of Dermal Microvascular Endothelial Cells in Psoriasis

Psoriasis is an inflammatory disease characterized by the abnormal proliferation of skin cells, including dermal microvascular endothelial cells. Recently, chemokine-like factor 1 (CKLF1) was found to participate in the local inflammation and cell proliferation. To explore its role in the pathogenesis of psoriasis, the expression of both CKLF1 and its receptor (CCR4) was determined in the psoriatic lesions. Also, the effect of the C-terminal peptides (C19 and C27) of CKLF1 on the proliferation of human umbilical vein endothelial cells was studied in vitro. By immunohistochemistry and immunofluorescence, the expression of both CKLF1 and CCR4 was determined in the psoriatic lesions. The effect of C-terminal peptides on human umbilical vein endothelial cells (HUVECs) was studied in vitro by the evaluation of cell proliferation and apoptosis. The in vivo assessment was performed accordingly through the subcutaneous injection peptides on BALB/c mice. The results showed that, by immunohistochemistry, both CKLF1 and CCR4 were increasingly expressed in psoriatic lesions as compared to normal skins. Moreover, the primary umbilical vein endothelial cells exhibited higher proliferation ratio under the C19 or C27 stimulation, which was even enhanced by the addition of psoriatic sera or TNF-α. Furthermore, the enhancement of peptide simulation was accompanied with the activation of ERK1/2-MAPKs pathway. In addition, such effect of C19 and C27 was mirrored by the hyperproliferation of cutaneous microvessels in BALB/c mice that were subcutaneously injected with the two peptides. Therefore, we concluded that CKLF1 plays a role in the pathogenesis of psoriasis by promoting the proliferation of microvascular endothelial cells that possibly correlates with ERK1/2-MAPKs activation.     

Selective serotonin reuptake inhibitors attenuate the antigen presentation from dendritic cells to effector T lymphocytes

Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), has been found to possess immune modulation effects, in addition to its antidepressant effects. However, it remains unclear whether SSRIs can suppress the antigen-presenting function of dendritic cells (DCs). Therefore, Fluoxetine was applied to a co-culture of Aggregatibacter actinomycetemcomitans (Aa)-reactive T cells (×Aa-T) isolated from Aa-immunized mice and DCs. This resulted in the suppressed proliferation of ×Aa-T stimulated with Aa-antigen presentation by DCs. Specifically, Fluoxetine increased the extracellular 5-hydroxytryptamine (5-HT) in the ×Aa-T/DC co-culture, whereas exogenously applied 5-HT promoted T-cell proliferation in the ×Aa-T/DC co-culture, indicating that Fluoxetine-mediated suppression of ×Aa-T/DC responses cannot be attributed to extracellular 5-HT. Instead, Fluoxetine remarkably suppressed the expression of costimulatory molecule ICOS-L on DCs. Fluoxetine also promoted a greater proportion of CD86(Low) immature DCs than CD86(High) mature DCs, while maintaining the expression levels of CD80, MHC-class-II and PD-L1. These results suggested that Fluoxetine suppressed the ability of DCs to present bacterial antigens to T cells, and the resulting T-cell proliferation, in a SERT/5-HT-independent manner and that diminished expression of ICOS-L on DCs and increase of CD86(Low) immature DCs caused by Fluoxetine might be partially associated with Fluoxetine-mediated suppression of DC/T-cell responses.     

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

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

Galectin-9 induces maturation of human monocyte-derived dendritic cells

Maturation of dendritic cells (DCs) is critical for initiation of immune responses and is regulated by various stimulatory signals. We assessed the role of galectin (Gal)-9 in DC maturation. Culture of immature DCs with exogenous Gal-9 markedly increased the surface expression of CD40, CD54, CD80, CD83, CD86, and HLA-DR in a dose-dependent manner, although Gal-9 had no or little effect on differentiation of human monocytes into immature DCs. Gal-9-treated DCs secreted IL-12 but not IL-10, and they elicited the production of Th1 cytokines (IFN-gamma and IL-2) but not that of the Th2 cytokines (IL-4 and IL-5) by allogeneic CD4+ T cells. These effects of Gal-9 on immature DCs were not essentially dependent on its lectin properties, given that they were inhibited only slightly by lactose. We further found that a Gal-9 mutant that lacks beta-galactoside binding activity reproduced the above activities and that an anti-Gal-9 mAb suppressed them. Gal-9 induced phosphorylation of the MAPK p38 and ERK1/2 in DCs, and an inhibitor of p38 signaling, but not inhibitors of signaling by either ERK1/2 or PI3K, blocked Gal-9-induced up-regulation of costimulatory molecule expression and IL-12 production. These findings suggest that Gal-9 plays a role not only in innate immunity but also in acquired immunity by inducing DC maturation and promoting Th1 immune responses.