Effects of levamisole on spontaneous rosette-forming cells in murine spleen.

Levamisole, an anti-anergic chemotherapeutic agent, is shown to depress the azathioprine sensitivity of rosette-forming cells (RFC) in the spleen of normal mice, and to restore the azathioprine sensitivity of RFC in the spleen of adult thymectomized mice. Using the fully active dose of 1.25 mg/kg i.v., the restoration effect was present 15 h after treatment with the drug and almost disappeared after 48 h. Levamisole was ineffective on RFC in vitro. These results suggest that Levamisole might interfere with the reactivity of T lymphocytes as has been described for drugs interfering with cyclic nucleotide metabolism.     

Anamnestic chemiluminescence of murine spleen cells

The oxidative response of murine spleen cells to secondary exposure to antigen was determined by luminol (5-amino-2,3-dihydro-1,4-pthalazinedione) amplified chemiluminescence, CL. BALB/cj and CBA/J mice were immunized with saline or an antigen solution of saline, luminol, and bovine serum albumin. Spleen cells were obtained from mice two and four days after immunization, and the CL response to in vitro antigenic exposure was measured for 35 minutes. At two days post-immunization, there was no difference in the CL of control and antigen-primed cells. By day four, the antigen-primed CL response differed significantly in both magnitude and time course from the primary antigen-stimulated response of the controls. This early development of differential CL response to antigenic challenge suggests a role for oxidative metabolic activity in the expression of the anamnestic immune response.     

Identification of a spleen cell-derived factor that inhibits sensitization of murine peritoneal mast cells as interferon-gamma

We present evidence that a spleen cell-derived factor that inhibits the sensitization of mouse peritoneal mast cells is IFN gamma. Conditioned medium (CM) from Con A-activated mouse spleen cells and recombinant MuIFN gamma both inhibited antigen-induced 5-HT release from peritoneal mast cells when added at the sensitization stage, but were without effect on presensitized cells. Both preparations were active at dilutions corresponding to similar levels of IFN activity (1-10 units/ml). The inhibitory activity of CM was blocked by a rat monoclonal MuIFN gamma-neutralizing antibody, thus confirming that IFN gamma was the active molecule.     

Conditioned medium from concanavalin A-stimulated spleen cells inhibits the IgE-dependent sensitization of murine peritoneal mast cells in vitro.

Conditioned medium (CM) from concanavalin A (Con A)-stimulated murine spleen cells inhibited release of histamine and 5-HT from murine peritoneal mast cells sensitized with monoclonal IgE anti-DNP antibody and challenged with DNP-human serum albumin (HSA) antigen. Inhibition was seen when the CM was added to the mast cells either 24 hr before or simultaneous with, but not 24 hr subsequent to, the IgE, thus showing that inhibition was at the IgE-dependent stage of mast cell sensitization. Unconditioned medium, prepared in the same way as CM but not exposed to spleen cells was without activity, demonstrating that inhibition was due to a spleen cell-derived factor. CM from unstimulated spleen cells was likewise without activity. The sensitization inhibitory factor appears to be a protein, since it was retained upon dialysis, and destroyed by heating at 70 degrees and above. The factor does not appear to be IgE, since it was stable at 56 degrees, and is not IL-1 or IL-2, since recombinant human IL-1 alpha and IL-1 beta, and recombinant mouse IL-1 alpha and IL-2 were without inhibitory activity. The active CM and all recombinant IL-1 and IL-2 preparations did not release histamine or 5-HT directly from mast cells during 48 hr of culture, and did not modulate the histamine content of these cells, nor their capacity to incorporate [3H]5-HT.     

ROS对小鼠腹腔巨噬细胞凋亡的影响

目的 探讨ＲＯＳ影响巨噬细胞凋亡的机制。方法 激光扫描共聚集显微术,流式细胞术和荧光标记技术等,结果（１）凋亡巨噬细胞内ＤＡＤＰＨ氧化酶活性急剧降低使得胞内ＲＯＳ水平快速上降;（２）ＲＯＳ清除剂促进地塞米松诱导的巨噬细胞凋亡;（３）ＰＫＣ促进巨细胞凋亡和ＲＯＳ急剧减少;ｃＡＭＰ抑制巨噬细胞凋亡和ＲＯＳ急剧减少。结论 （１）ＲＯＳ抑制地塞米松诱导的巨噬细胞凋亡;（２）ＰＫＣ,ｃＡＭＰ等因素通过影响地     

Effects of irradiation upon the response of murine spleen cells to mitogens.

The mitogenic response of murine spleen cells exposed to graded doses of radiation was evaluated. Low-dose exposures were associated with an augmented response to concanavalin A (Con A) that was most marked with 100 rads. Low-dose augmentation of phytohemagglutinin (PHA) stimulation was equivocal and most pronounced in cells exposed to 10-20 rads. Augmentation was only demonstrable when the cells were irradiated immediately prior to mitogenic stimulation. Timed exposures after stimulation with Con A or PHA showed no evidence that mitogen activation increased radioresistance, although the possibility could not be excluded that activation protects against interphase cell death. Reduced isotope incorporation was associated with all doses of radiation evaluated in cells stimulated with lipopolysaccharide (LPS) or pokeweed mitogen (PWM). On this basis it is concluded that 1) each of the mitogens tested differs in its capacity to stimulate irradiated spleen cells; 2) radiation-induced augmentation is noted with those mitogens (Con A and possibly PHA) known to activate only T cells; 3) radiation-induced augmentation may be due to the release of mitogenically active molecules by injured lymphocytes.     

Fusion of murine myeloma cells with murine spleen cells: A simple method

Summary A simple and effective method is described for produeing hybrid cells from the fusion of parental murine myeloma cells with spleen cells from an immunized mouse. This method results in minimal damage to the fused cells.     

杂色曲霉素对小鼠脾细胞IL-4 mRNA表达和蛋白分泌的影响

目的：探讨杂色曲霉素（ST）对体外培养的小鼠脾细胞IL-4 mRNA表达及其蛋白分泌的影响。 方法： 分别采用半定量RT-PCR及ELISA方法，研究5种不同剂量ST（0.125 mg/L、0.25 mg/L、0.5 mg/L、1 mg/L、2 mg/L）预处理2 h、12 h对小鼠脾细胞IL-4 mRNA表达及其蛋白分泌的影响，观察ST对IL-4影响的时效及量效关系。 结果： ST预处理2 h，较小剂量ST（0.125、0.25、0.5 mg/L）处理组小鼠脾细胞IL-4 mRNA的表达高于对照组，以0.5 mg/L组表达最高；当ST预处理达到12 h时，较小剂量ST处理组IL-4 mRNA表达均低于对照组，其中以ST 0.125、0.25 mg/L组降低最明显。而大剂量ST（1 mg/L、2 mg/L）处理2 h及12 h对小鼠脾细胞IL-4 mRNA表达均低于对照组。在蛋白水平上的改变与mRNA水平变化相似。 结论： ST对小鼠脾细胞IL-4 mRNA表达的影响与其剂量和作用时间有关，既可表现为抑制作用，也可表现诱导作用。     

杂色曲霉素对小鼠脾细胞IL-2及IFN-γ分泌和表达的影

目的探讨杂色曲霉素(ST)对体外培养的小鼠脾细胞IL-2及IFN-γ mRNA表达及其蛋白分泌的影响.方法分别采用半定量RT-PCR及ELISA方法,研究5种不同剂量ST(0.125 mg/L,0.25 mg/L,0.5 mg/L,1 mg/L,2 mg/L)预处理对小鼠脾细胞IL-2及IFN-γ mRNA表达及其蛋白分泌的影响.结果不同剂量ST预处理2 h均可引起小鼠脾细胞IL-2及IFN-γ表达的改变,但不同剂量影响不同,小剂量ST处理组(0.125、0.25 mg/L)在ST处理后2 h,可诱导脾细胞IL-2及IFN-γ mRNA的表达;而大剂量ST处理组(1 mg/L、2 mg/L)可明显抑制脾细胞IL-2及IFN-γ mRNA表达及其相应蛋白的分泌,尤以ST 1 mg/L的抑制作用最明显.结论ST可影响小鼠脾细胞IL-2及IFN-γ表达和分泌的改变,较小剂量组(0.125、0.25 mg/L)表现为诱导作用,而较大剂量组(1 mg/L、2 mg/L)则表现为明显抑制作用.     

Vilon(L-Lys-L-Glu)对小鼠腹腔巨噬细胞分泌IL-1β、NO的影响

目的 探讨Vilon(L-Lys-L-Glu)对参与炎症反应的小鼠腹腔巨噬细胞分泌IL-1β、NO的影响.方法 以小鼠原代培养腹腔巨噬细胞为阳性参照,ELISA法检测Vilon及脂多糖(LPS)共刺激的小鼠腹腔巨噬细胞IL-1β的分泌水平;还原酶法分析NO分泌水平;RT-PCR法检测IL-1β和iNOS mRNA的表达.结果 Vilon和LPS共刺激小鼠腹腔巨噬细胞时,Vilon对LPS活化的小鼠腹腔巨噬细胞分泌IL-1β及NO具有明显的促进作用,并且呈剂量依赖关系;同时也促进了IL-1β和iNOS mRNA表达.结论 Vilon对活化的小鼠腹腔巨噬细胞分泌IL-1β、NO具有明显的促进作用.     

高糖腹膜透析液对小鼠腹腔巨噬细胞活力和NO产生的影响

目的探讨商业用腹膜透析液(commercial peritoneal dialysate,CDS)对腹腔巨噬细胞功能的影响.方法培养的小鼠腹腔巨噬细胞(Mφ)在含不同葡萄糖浓度(15、25和42 g/L)的CDS中,经不同时间(10、30和60 min)的暴露,观察MФ还原MTF的能力及其NO的产量.结果低葡萄糖浓度(25 g/L)暴露10 min实验组,MФ还原MTT的能力(A570nm值)为0.210±0.008,NO的产量为(9.1±1.3)μmol/L,均明显低于对照组(P＜0.01);高葡萄糖浓度(42 g/L)和较长时间(60 min)暴露组,MФ还原MTF的能力(A570nm值)为0.056±0.004,NO的产量为(5.7±1.1)μmol/L,与对照组相比较,改变最明显(P＜0.01).结论短时间CDS暴露,即可降低MФ的活力及NO产生,这种作用与CDS中的葡萄糖浓度以及MФ暴露于CDS的时间呈正相关.     

Simultaneous expression of multiple immune complex receptors on murine thymocytes and spleen cells

Mixed rosette studies were performed to evaluate the coexpression of IgG Fc. IgM Fc, and complement receptors (C3R) by thymocytes obtained from mice 7 days after cortisone injection and by spleen cells. Indicator cells coated with IgM, IgG, or C3 independently were mixed and could be distinguished by morphology or by a fluorescein label. In double-marker studies, 36% of spleen cells formed rosettes with IgG- and/or IgM-sensitized red blood cells. Among this population there was a 24% overlap of cells binding IgM and IgG complexes simultaneously. Of the spleen cells, 78% bound IgM- and/or C3-sensitized cells. Of the spleen cells forming rosettes with IgM and C3 indicator cells, 15% coexpressed these receptors. With IgG and C3 indicator cells, 58% of spleen cells bound to one or both kinds of complexes with an 18% overlap. Of cortisone-resistant thymocytes, 14% formed rosettes with IgM- and/or IgG-sensitized red blood cells; within this population there was an overlap of 21%. With IgM- or C3-sensitized cells, 19% of cortisone-resistant thymocytes bound to one or both, among which there was a coexpression of 21%. With IgG- or C3-sensitized cells, there was a 14% overlap of rosette-forming cells binding both. In triple-marker studies 79% of spleen cells formed rosettes with C3-, IgG-, and/or IgM-sensitized indicator cells, out of which 11% coexpressed IgM and IgG FcR, 20% coexpressed IgG and C3R, and 10% coexpressed IgM FcR and C3R. Of rosette-forming cells, 13% coexpressed all three receptors. With cortisone-resistant thymocytes, 19% bound one or more kinds of immune complexes. Among these, 9% coexpressed IgG FcR and C3R, 14% coexpressed IgM FcR and C3R, and 14% bound IgG and IgM complexes. We could not detect the simultaneous expression of all three receptors on cortisone-resistant thymocytes. Using Isopaque-Ficoll fractionation of cells binding C3-sensitized cells, cortisone-resistant thymocytes were enriched and depleted of C3-receptor-bearing cells and their Lyt phenotypes were determined by immunofluorescence microscopy. The C3-receptor-enriched population contained 56% C3R+ cells which were 79% Lyt-1 positive and 100% Lyt-2 positive. The C3R-depleted population contained 1.3% C3R+ cells with 10% Lyt-1 positive and 22% Lyt-2 positive among the total. Surface phenotypic expression of normal and cortisone-resistant thymocytes was also evaluated by direct and indirect fluorescence by fluorescence-activated cell sorter (FACS).(ABSTRACT TRUNCATED AT 400 WORDS)     

人参皂苷Rg1对小鼠腹腔巨噬细胞的影响

目的研究人参皂苷Rg1对小鼠腹腔巨噬细胞的影响,并探讨其作用机制。方法无菌分离小鼠腹腔巨噬细胞,制备单细胞悬液,加入不同终浓度的人参皂苷Rg1 4 h后,细菌脂多糖LPS(10μg/ml)作用24 h,直径1μm的微球(1×1010/L)结合流式细胞术分析Rg1对小鼠腹腔巨噬细胞吞噬作用的影响;Griess试剂盒检测NO的释放;H2DCFDA染色检测ROS的含量;Fluo-4/AM染色检测Rg1对Ca2+超载的影响;Sytox R Green染色,荧光酶标仪检测Rg1对CHX、CTX诱导的细胞凋亡的影响。结果 10、20μmol/L的Rg1时能明显抑制LPS诱导的巨噬细胞NO和ROS的产生以及吞噬微球的能力;10μmol/L的Rg1能显著抑制Ion诱导的的巨噬细胞Ca2+的超载以及CHX、CTX诱导的细胞的凋亡。结论 Rg1具有显著的抗炎作用,并可抵抗多种因素诱导的细胞凋亡,为进一步开发为免疫治疗药物提供了依据。     

鼠脾细胞肿瘤坏死因子的诱生和检测

本文报道采用Ｅ．ｃｏｌｉ ＬＰＳ刺激ＡＬＢ／Ｃ小鼠脾细胞诱生ＴＮＦ的最适条件，并用人ＴＮＦ ＥＬＩＳＡ方法检测小鼠ＴＮＦ。结果显示：脾细胞经ＬＰＳ刺激２ｈ就可在上清液中测出ＴＮＦ，１２ｈ达高峰，ＬＰＳ最适刺激浓度为１０＾－２μｇ／ｍｌ，ＴＮＦ产量依赖于脾细胞的浓度。     

Detection of a membrane-like IgM inside of murine spleen cells

In an effort to determine at what stage during intracellular transport the precursor to plasma membrane IgM becomes inserted in membranes, the solubility (in the absence of detergent) of radioactive IgM obtained by incubation of cells with³H-leucine was examined. A population of normal spleen cells of mice were used since a considerable proportion of the IgM biosynthesized is membrane IgM and because synthesis of other Ig classes is minimal. Detergent lysates of labeled cells were treated with Bio-Beads SM-2 to remove the detergent, Nonidet P40. Large aggregates were then separated from smaller aggregates and soluble molecules by ultracentrifugation. Between 30 and 40% of the³H-IgM was associated with the large aggregate fraction. Mouse IgG and partially-reduced MOPC 104E mouse IgM did not aggregate under these conditions. Aggregation of pentameric MOPC 104E IgM was sensitive to detergent. Fetal calf serum at 10% concentration reduced aggregation. The percentage of³H-IgM in the large aggregate fraction did not vary with the length of incubation of cells in the presence of radioactive amino acid between 15 min and 2 hr. The ability of intracellular or plasma membrane Ig (labelled with¹²⁵I) to aggregate was not sensitive to prior reduction of the inter-heavy chain bonds. The results are interpreted as supporting the view that insertion into a lipid bilayer is an early event in the biosynthesis of membrane IgM. It was also found that intracellular IgM shares with membrane IgM an anomalously slow mobility on electrophoresis in polyacrylamide gels containing dodecyl sulfate and that the anomalous mobility was no longer associated with molecules that had been partially reduced.     

Novel rosette formation of murine spleen cells with autologous red blood cells

Spleen cells of normal BALB/c mice formed rosettes with autologous red blood cells, and the formation was calcium ion dependent. Peritoneal exudate cells, bone marrow cells and thymocytes did not form such rosettes. Spleen cells were passed over a Sephadex G-10 column or incubated on a plastic surface in order to eliminate adherent cells from them. Cells obtained by both these methods were unable to form rosettes. B cell-, T cell- and natural killer cell-enriched fractions in spleen cells were unable to form rosettes either. Some of the mouse IgG subclasses suppressed rosette formation when added to its site. These are monoclonal antibodies whose specificities are directed against Aspergillus niger glucose oxidase. Moreover, Aspergillus niger glucose oxidase suppressed the rosette formation when spleen cells had been treated with it as well as when it had been added to the site of rosette formation. These findings suggest that some murine spleen cells have receptors to a structure on autologous red blood cells, which is recognized by an anti-Aspergillus niger glucose oxidase monoclonal antibody.     

Growth of Trypanosoma musculi in cultures of murine spleen cells and analysis of the requirement for supportive spleen cells.

Growth of Trypanosoma musculi in vitro has been achieved. The number of parasites increased by more than 1,500-fold in less than 8 days under the most suitable conditions. The rate and magnitude of growth was comparable to that which occurs in inoculated murine hosts. Maximum growth was displayed in cultures composed of RPMI 1640 medium supplemented with fetal calf serum, murine spleen cells, and foreign erythrocytes (sheep). No growth occurred in the absence of spleen cells. The adherent, macrophage-rich population supported parasite growth much better than did the nonadherent population. Parasite growth was excellent in the presence of irradiated spleen cells or of cells from thymectomized, irradiated, bone marrow-reconstituted mice. The important cells appeared to be macrophages. The beneficial effect of sheep erythrocytes probably resulted from preoccupation or stimulation of phagocytes. Soluble substances released by spleen cell cultures promote parasite growth, as was shown by experiments with double-compartment culture vessels. The utility of this culture system for analysis of host immune responses against the trypanosome was demonstrated.     

Analysis of the CCR7 expression on murine bone marrow-derived and spleen dendritic cells

About 40% of bone marrow-derived dendritic cells (BM-DCs) generated from stem cells of C57BL/6 (B6.WT) mice differentiate in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) without further stimuli to mature DCs. These cells are characterized by high levels of major histocompatibility complex class II, CD40, and CD86 on their surface. Recent studies have revealed that tumor necrosis factor (TNF) is crucial for maturation of BM-DCs. However, once matured, the phenotype of mature TNF-negative C57BL/6 (B6.TNF-/-) and B6.WT BM-DCs is comparable. Both expressed high levels of CD40 and CD86 and were positive for mRNA of the chemokine receptor (CCR)7. To extend our studies, we generated a monoclonal antibody (mAb) specific for mouse CCR7. This mAb allowed us to analyze the surface expression of CCR7 during maturation of B6.WT and B6.TNF-/- BM-DCs in the presence of GM-CSF and stimulated with TNF or lipopolysaccharide (LPS) and to compare it with the CCR7 expression on ex vivo-isolated splenic DCs with or without additional stimulation. Our results showed that CCR7 expression on murine BM-DCs is an indication of cell maturity. Incubation with LPS induced the maturation of all BM-DCs in culture but increased the number of mature CCR7+ splenic DCs only marginally.