肿瘤微环境对树突细胞功能状态的影响

目的：探讨肿瘤细胞分泌的可溶性细胞因子营造的微环境对树突细胞(DCs，dendritic cells)的分化发育的影响，以进一步揭示肿瘤的免疫逃逸机制。方法：用免疫磁珠从人外周血分离CD14^ 单核细胞，加入粒细胞巨噬细胞集落刺激因子(GM-CSF)、白细胞介素4(IL-4)和人白血病细胞Jurkat培养上清液体外培养DCs，以正常培养诱导的DCs作为对照，采用傅立叶变换红外光谱(FTIR)技术分析人白血病细胞培养上清液对DCs分化发育的影响。结果：正常培养DCs与肿瘤上清液培养的DCs相比，在细胞内蛋白质和核酸的相对含量和细胞内消耗葡萄糖重新合成磷脂的量方面差异无显著性，但是，在细胞的转录状态方面差异有显著性。结论：肿瘤细胞上清液培养液所营造的微环境细胞转录水平上对DCs的功能状态有明显的抑制作用。     

人外周血单核细胞和脐血CD34+细胞来源的树突细胞比较

目的比较从人外周血单核细胞及脐血CD34+细胞诱导树突细胞(DC)的方法及其特性.方法用淋巴细胞分离液分离外周血单个核细胞,贴壁法获得单核细胞;经GM-CSF及IL-4诱导获得DC.磁性活化细胞分选系统(MACS)分选脐血CD34+细胞,以GM-CSF、IL-4、TNF-α、SCF、FL、TPO等细胞因子诱导获得DC.用电镜、普通显微镜观察DC形态;流式细胞仪分析DC表型;同种混合淋巴细胞反应(allo-MLR)观察DC刺激T淋巴细胞增殖的能力.结果外周血单核细胞在因子GM-CSF、IL-4、TNF-α,脐血CD34+细胞在GM-CSF、IL-4、TNF-α、SCF、FL、TPO等因子作用下,可生成DC,并表达相应的DC分化抗原CD14、CD80、CD83、CD86、HLA-DR.脐血CD34+细胞具有较强的扩增能力,经2周诱导,体系细胞数可增加173.8士26.7倍,两种来源的DC均能刺激同种异体淋巴细胞的增殖反应.结论外周血单核细胞与脐血CD34+细胞,在体外均可诱导成DC,并具有相应的DC分化抗原和功能.     

Interleukin 6 Influences Germinal Center Development and Antibody Production via a Contribution of C3 Complement Component

Mice rendered deficient for interleukin (IL) 6 by gene targeting were evaluated for their response to T cell–dependent antigens. Antigen-specific immunoglobulin (Ig)M levels were unaffected whereas all IgG isotypes showed varying degrees of alteration. Germinal center reactions occurred but remained physically smaller in comparison to those in the wild-type mice. This concurred with the observations that molecules involved in initial signaling events leading to germinal center formation were not altered (e.g., B7.2, CD40 and tumor necrosis factor R1). T cell priming was not impaired nor was a gross imbalance of T helper cell (Th) 1 versus Th2 cytokines observed. However, B7.1 molecules, absent from wild-type counterparts, were detected on germinal center B cells isolated from the deficient mice suggesting a modification of costimulatory signaling. A second alteration involved impaired de novo synthesis of C3 both in serum and germinal center cells from IL-6–deficient mice. Indeed, C3 provided an essential stimulatory signal for wild-type germinal center cells as both monoclonal antibodies that interrupted C3-CD21 interactions and sheep anti–mouse C3 antibodies caused a significant decrease in antigen-specific antibody production. In addition, germinal center cells isolated from C3–deficient mice produced a similar defect in isotype production. Low density cells with dendritic morphology were the local source of IL-6 and not the germinal center lymphocytes. Adding IL-6 in vitro to IL-6–deficient germinal center cells stimulated cell cycle progression and increased levels of antibody production. These findings reveal that the germinal center produces and uses molecules of the innate immune system, evolutionarily pirating them in order to optimally generate high affinity antibody responses.     

New metrics for analysis of dendritic branching patterns demonstrating similarities and differences in ON and ON-OFF directionally selective retinal ganglion cells.

The morphology and dendritic branching patterns of retinal ganglion cells have been studied in Golgi-impregnated, whole-mount preparations of rabbit retina. Among a large number of morphological types identified, two have been found that correspond to the morphology of ON and ON-OFF directionally selective (DS) ganglion cells identified in other studies. These two kinds of DS ganglion cell are compared with each other, as well as with examples of class I, class II, and class III cells, defined here with reference to our previous studies. Cell body, dendritic field size and branching pattern are analyzed in this paper and levels of dendritic stratification are examined in the following paper. ON DS ganglion cells are about 10% larger in soma size and about 5 times the dendritic field area of ON-OFF DS ganglion cells, when compared at the same retinal location. These two morphological types of ganglion cell can be said to define the upper and lower bounds of an intermediate range of cell body and dendritic field sizes within the whole population of ganglion cells. Nevertheless, in previous physiological studies receptive field sizes of the two types were shown to be similar. This discrepancy between morphological and physiological evidence is considered in the Discussion in terms of a model of the excitatory receptive field of ON-OFF DS ganglion cells incorporating starburst amacrine cells. A new set of metrics is introduced here for the quantitative analysis and characterization of the branching pattern of neuronal arborizations. This method compares the lengths of terminal and preterminal dendritic branches (treated separately), as a function of the distances of their origins from the soma, viewed graphically in a two-dimensional scatter plot. These values are derived from computer-aided 3D logging of the dendritic trees, and distance from the soma is measured as the shortest distance tracked along the dendritic branches. From these metrics of the "branch length distributions," scale-independent branching statistics are derived. These make use of mean branch lengths and distances, slopes of lines fitted to the distributions, and elliptical indices of scatter in the distributions. By these measures, ON and ON-OFF DS ganglion cells have similar branching patterns, which they share to varying degrees with functionally unrelated class III.1 ganglion cells. The scale of the branching patterns of ON and ON-OFF DS cells and their degree of uniformity are different, however.(ABSTRACT TRUNCATED AT 400 WORDS)     

Morphology of retinal ganglion cells in lungless salamanders (fam. Plethodontidae): an HRP and Golgi study

The family Plethodontidae consists of nearly two-thirds of all living urodeles; most of them possess highly developed visual abilities. We investigated the morphology of retinal ganglion cells (RGCs) in four representative species by means of the horseradish peroxidase method in flatmounts and in transverse sections and with the Golgi method in transverse sections. In flatmount preparations, four classes of RGCs were found, differing in dendritic arborization, dendritic field size, and stratification pattern of dendrites in the inner plexiform layer (IPL). Class-1 cells had small dendritic fields (29-44 microns 2) and arborized throughout the entire depth of the IPL. Class-2 cells had medium to large dendritic fields (75-206 microns 2) and mostly arborized in two or three laminae or in a diffuse fashion in the IPL. Class-3 cells had medium to large dendritic fields (72-200 microns 2) but sparse dendritic arborization. They only arborized in the proximal lamina of the IPL. Class-4 cells had large dendritic fields (273-626 microns 2) and branched in the most sclerad stratum of the IPL. No large differences in intraspecific soma size of the different RGC classes were detected (although interspecific soma size varied to a considerable degree) and no "giant" cells typically found in other vertebrate retinas were present. The results suggest that, with respect to the pattern of arborization and stratification of dendrites, lungless salamanders possess morphological classes of RGC similar to those found in frogs, but the morphology of RGCs in lungless salamanders seems to be simplified in comparison to frog RGCs. This simplification might be a consequence of paedomorphosis.     

Osteopontin affects the persistence of beta-glucan-induced hepatic granuloma formation and tissue injury through two distinct mechanisms

Osteopontin (OPN) plays a pivotal role in various immune responses and inflammatory diseases. OPN is expressed in various granulomatous diseases; however, the cellular and molecular role of OPN in these diseases is not well known. We analyzed the role of OPN in a beta-glucan-induced hepatic granuloma model. First, we found that neither OPN deficiency nor overexpression of OPN affected the number and the size of hepatic granulomas at day 7, indicating that OPN is not involved in the formation of hepatic granulomas at the early stages. Importantly, OPN did not influence the liver tissue damage as defined by alanine aminotransferase and aspartate aminotransferase levels at early stages. Second, OPN deficiency resulted in the reduction of IL-12 and IFN-gamma production at early stages. Third, at late stages, OPN deficiency resulted in a decrease in the number and size of hepatic granulomas, and a reduction of liver tissue injury. This was due to the reduction of the cellular recruitment including macrophages, CD4 T cells and dendritic cells into the liver, and the reduction of tumor necrosis factor (TNF)-alpha production in the liver. In contrast, overexpression of OPN resulted in the persistence of granuloma formation. These data suggest that OPN affects the persistence of hepatic granuloma formation. Our results indicate that OPN up-regulates the production of IL-12 and IFN-gamma within the granulomas at early stages, and OPN has an additional role in the regulation of cellular recruitment and TNF-alpha production at late stages that determine the severity of liver tissue injury.     

IDO expression on decidual and peripheral blood dendritic cells and monocytes/macrophages after treatment with CTLA-4 or interferon-gamma increase in normal pregnancy but decrease in spontaneous abortion

Recent data demonstrated that CD4+CD25+ regulatory T (Treg) cells and an enzyme called indoleamine 2,3-dioxygenase (IDO) mediate maternal tolerance to the fetus. Interestingly, Treg cells express the CTLA-4 molecule on their surface, and B7 (CD80/86) ligation by CTLA-4 enhanced IDO activity of dendritic cells (DCs) and monocytes by the induction of interferon gamma (IFN-gamma) production. In this study, we studied the IDO expression on peripheral blood monocytes and decidual monocytes or DCs after treatment with CTLA-4/Fc fusion protein or IFN-gamma using flow cytometry. IDO expressions on both peripheral blood DC and decidual DC and monocytes were up-regulated during normal pregnancy. On the other hand, both IDO expression on DC and monocytes after IFN-gamma treatment or CTLA-4 treatment were decreased in spontaneous abortion cases. The expression of CD86 on peripheral blood and decidual monocytes and DC in spontaneous abortion cases was lower compared with those in normal pregnancy subjects. Also, IFN-gamma production by decidual and peripheral blood mononuclear cells after CTLA-4/Fc treatment in spontaneous abortion cases was significantly lower than those in normal pregnancy subjects. These data suggest that CTLA-4 on Treg cells up-regulates IDO expression on decidual and peripheral blood DC and monocytes by the induction of IFN-gamma production.     

Effect of ozone exposure on allergic sensitization and airway inflammation induced by dendritic cells

BACKGROUND: Epidemiological studies suggest that ozone exposure is related to increased asthma symptoms. Dendritic cells (DCs) are the principal antigen-presenting cells in the airways. OBJECTIVE: We have examined whether ambient doses of ozone (100 ppb for 2 h) enhance allergic sensitization and/or airway inflammation in a mouse model. METHODS: C57BL/6 mice were sensitized to inhaled ovalbumin (OVA) by intratracheal instillation of OVA-pulsed DCs on day 0. Daily exposure to OVA aerosol on days 14-20 resulted in an eosinophilic airway inflammation, as reflected in bronchoalveolar lavage fluid and lung histology. In a first experiment, mice were exposed to ozone or room air immediately prior to and following sensitization. Subsequently, we tested the effect of ozone exposure during antigen challenge in DC-sensitized mice. RESULTS: Exposure to ozone during sensitization did not influence airway inflammation after subsequent allergen challenge. In contrast, in sensitized mice, challenge with OVA together with ozone (days 14-20) resulted in enhanced airway eosinophilia and lymphocytosis, as compared with mice exposed to OVA and room air (1.91 x 106 +/- 0.46 x 106 vs. 0.16 x 106 +/- 0.06 x 106 eosinophils/mL lavage fluid; P = 0.015; 0.49 x 106 +/- 0.11 x 106 vs. 0.08 x 106 +/- 0.03 x 106 lymphocytes/mL lavage fluid; P = 0.004). Ozone exposure without subsequent OVA exposure did not cause airway inflammation. CONCLUSION: Ozone exposure does not increase allergic sensitization but enhances antigen-induced airway inflammation in mice that are sensitized via the airways.     

Ligand and cytokine dependence of the immunosuppressive pathway of tryptophan catabolism in plasmacytoid dendritic cells

Murine plasmacytoid dendritic cells (pDCs) have been credited with a unique ability to express indoleamine 2,3-dioxygenase (IDO) function and mediate immunosuppression in specific settings; yet, the conditions of spontaneous versus induced activity have remained unclear. We have used maneuvers known to up-regulate IDO in different cell types and have examined the relative efficacy and mechanisms of the induced activity in splenic pDCs, namely, after specific receptor engagement by CTLA-4-Ig, CD200-Ig or CD28-Ig, the latter in combination with silenced expression of the suppressor of cytokine signaling 3 (SOCS3) gene. We found that pDCs (CD11c+ mPDCA-1+ 120G8+) do not express IDO and are not tolerogenic under basal conditions. B7-1 engagement by CTLA-4-Ig, CD200R1 engagement by CD200-Ig and B7-1/B7-2 engagement by CD28-Ig in SOCS3-deficient pDCs were each capable of initiating IDO-dependent tolerance via different mechanisms. IFN-gamma was the major cytokine responsible for CTLA-4-Ig effects, and type I IFNs for those of CD200-Ig. Immunosuppression by CD28-Ig in the absence of SOCS3 required IFN-gamma induction and IFN-like actions of IL-6. Therefore, although pDCs do not mediate IDO-dependent tolerance constitutively, multiple ligands and cytokines will contribute to the expression of a tolerogenic phenotype by pDCs in the mouse.     

Mechanisms Involved in the Binding of Thymocytes to Rat Thymic Dendritic Cells

The effects of monoclonal antibodies (mAbs) to cell-surface molecules, divalent cations, and various cell-signaling and metabolic inhibitors on the binding of thymocytes to rat thymic dendritic cells (TDC) were studied using a rosette assay. It was found that TDC/thymocyte adhesion was stronger and faster at 37°C than at 4°C. Flow cytometric analysis demonstrated that bound thymocytes were predominantly CD4⁺CD8⁺ and CD4⁺CD8^-, but in comparison to the phenotype of whole thymocytes, they were enriched in the mature TCRαβ^hi subset. The binding of thymocytes to TDC at 37°C was almost completely dependent on Ca²⁺ and Mg²⁺ and partly on an intact cytoskeleton and calmodulin-dependent protein kinase. The adhesion was independent of new protein synthesis and the activities of protein kinases A and C, tyrosine kinases, as well as phosphotyrosine protein phosphatases. The TDC/thymocyte adhesion at 37°C was partly blocked by anti-LFA-1 (WT.1), anti-CD18 (WT.3), and anti-ICAM-1 (1A29) mAb. MAbs to class II MHC (OX-3 and OX-6), CD4 (W3/25), CD8 (OX-8), and αβTCR (R73) stimulated the adhesion via an LFA-1-dependent pathway, whereas an anti-CD45 mAb (G3C5) stimulated the rosette formation independently of LFA-1. MAbs to CD2 (OX-34), CD11b (ED7), CD11b/c (OX-42), and class I MHC (OX-18) were without significant effects on the adhesion process.