维生素D3诱导的U937细胞中CD14表达的实验研究

目的: 探讨维生素D3诱导U937细胞上CD14蛋白的表达及其对内毒素 (LPS)刺激的反应性。方法: 用0. 1μmol/LVitD3与U937细胞共同培养 24h诱导CD14基因的表达, 并观察U937细胞对不同浓度的LPS刺激不同时间的反应性。结果: VitD3能稳定诱导U937细胞表达CD14mRNA和CD14蛋白。经VitD3诱导的U937细胞对LPS刺激的敏感性显著增强, 表现为低浓度LPS刺激即能诱导该细胞核中NF -κB激活, 促进TNF- αmRNA的转录和表达, 并将表达的TNF α释放入培养上清中。结论: VitD3能诱导U937细胞中CD14基因和蛋白的表达, 并增加其对LPS刺激的反应性。     

rGM—CSF对U937细胞系膜MHC分子及CD86分子表达的作用

本实验研究了人重组ＧＭ－ＣＳＦ对Ｕ９３７人单核细胞样细胞系ＨＬＡ和ＣＤ８６分子表达的调节作用，将Ｕ９３７细胞在ｒＧＭ－ＣＳＦ１０μｇ／Ｌ培养１ｄ，ＨＬＡ－ＤＲ分子的表达率为８３％，对照组为９１％，培养ｄ表达率为５１％，对照组为７８％，培养５ｄ，表达率为５３％，对照组为８１％，Ｕ９３７细胞在ｒＧＭ－ＣＳＦ１０μｇ／Ｌ浓度下培养１ｄ，ＣＤ８６分子表达率为２％，对照组为１．６％，培养３ｄ，表达率为３．９     

TM-TNF-α介导的反向信号下调sTNF-α激活后U937细胞因子mRNA的表达

目的：观测TM-TNF-α介导的反向信号对sTNF-α激活后U937细胞因子mRNA的影响，构建TM-TNF-α胞浆段缺失突变体，并进一步验证。方法：sTNF-α激活U937细胞后撤除，加入可溶性TNFR（sTNFRⅠ）激活TM-TNF-α介导的反向信号，用RT—PCR方法检测反向信号对活化后U937细胞因子IL-1β、IL-8 mRNA的影响；采用分子克隆技术构建TM-TNF-α胞浆段缺失突变重组体，采用电穿孔法转染U937，并用Western blot检测蛋白表达。结果：sTNF-α激活U937细胞后撤除，高表达的细胞因子IL-1β、IL-8 mRNA随着时间的延长而逐渐降低；sTNFRⅠ激活的TM-TNF-α介导的反向信号可加速细胞因子mRNA的降解，且使mRNA降解至50％的时间分别由约75、60分钟缩短至约45、35分钟。成功构建TM-TNF—α胞浆段缺失突变重组体pcDNA3．0-△csTM-TNF—α并瞬时转染U937细胞，Western blot结果显示U937细胞膜表面除表达26000的野生型TM-TNF-α蛋白外，还可表达约20000的突变体蛋白。这种缺失胞浆段的突变体蛋白可竞争性抑制反向信号对活化后U937细胞因子mRNA的下调作用。结论：TM-TNF-α介导的反向信号可下调sTNF-α激活后U937细胞因子mRNA的表达；且TM-TNF-α胞浆段为其介导的反向信号所必需。     

ABCA1 mRNA expression and cholesterol outflow in U937 cells

Objective: To investigate the oxidized low density lipoprotein (oxLDL) on U937 cell ATP-binding cassette transporter A1 (ABCA1) mRNA expression and cholesterol efflux situation. Methods: Human U937 cells were incubated with gradient concentrations of oxLDL (0, 25, 50, 75, 100, 125 mg/L), and then dyed by oil red O to estimate the content of intracelluar lipid and detect the expressing quantity of ABCA1 mRNA by Real-time Fluorescence quantitative PCR simultaneously. Calculating the cholesterol efflux rates by using the scintillation counter to detect the amount of H³-cholesterol in each well cell culture plate and medium. Results: Real-time Fluorescence quantitative PCR analysis showed that the expression levels of ABCA1 mRNA in monocytes were lower than basal line when not intervened with oxLDL, and increased drastically with oxLDL stimulation, significant difference compared with controls (P < 0.01), and reached the highest level at oxLDL 50 mg/L, nevertheless, continuously increasing the concentration of oxLDL above 50 mg/L, the expression decreased. So is the outflowing rate of intracelluar lipid. Oil red O dyeing results also suggested that celluar lipid content was the highest when intervened with 125 mg/L oxLDL, and increased most obviously at 50 mg/L oxLDL. Cholesterol outflow result also demonstrated that cholesterol outflow rate related with the ABCA1 mRNA expressing quantity. Conclusion: With the increase of intervening concentration of oxLDL on U937cells, the exprssion of ABCAl mRNA represented that rising before 50 mg/L oxLDL, and then decreasing, reaching the top point at 50 mg/L oxLDL. So was the change in the outflowing rate of intracelluar lipid.     

LPS调节U937细胞上B7-H1表达的初步研究

目的了解脂多糖（LPS）刺激后U937细胞上B7-H1的表达变化情况。方法培养U937细胞,用荧光半定量实时PCR和流式细胞仪技术,分别观测未刺激和用LPS刺激的U937细胞B7-H1mRNA与蛋白的表达情况。结果未经刺激的U937细胞组成性表达B7-H1,LPS刺激可显著增强B7-H1基因mRNA的转录和蛋白的表达。结论LPS在转录与翻译两个环节均可上调U937细胞B7-H1的表达水平。     

Origins and functional basis of regulatory CD11c+CD8+ T cells

Previously, we showed that CD11c defines a novel subset of CD8⁺ T cells whose in vivo activity is therapeutic for arthritis; however, the mechanisms directing their development, identity of their precursors, and basis of their effector function remain unknown. Here, we show that the novel subset develops from CD11c^surface?CD8⁺ T cells and undergoes robust expansion in an antigen‐ and 4‐1BB (CD137)‐dependent manner. CD11c⁺CD8⁺ T cells exist in naïve mice (<3%) with limited suppressive activity. Once activated, they suppress CD4⁺ T cells in vivo and in vitro. Suppression of CD4⁺ by CD11c⁺CD8⁺ T cells is related to an increase in IDO activity induced in competent cells via the general control non‐derepressible‐2 pathway. CD11c⁺CD8⁺ T cells are refractory to the effect of IDO but constrict in a novel 1‐methyl D ,L ‐tryptophan‐dependent mechanism resulting in reversal of their suppressive effects. Thus, our data uncover, for the first time, the origin, development, and basis of the suppressive function of this novel CD11c⁺CD8⁺ T‐cell subpopulation that has many signature features of Treg.     

CD11c decrease in mouse thymic dendritic cells after vanadium inhalation

Vanadium (V) is a transition metal found in air adsorbed onto suspended particles. As a result, urban populations are often exposed to this element as a constituent of particulate matter (PM). One aspect of the myriad toxicities that might arise from these exposures is altered immune responses. Previous reports from the laboratory reported modifications in splenic architecture – with germinal center hyperplasia and a suppressed humoral immune response – in mice that had been exposed to vanadium agents via inhalation. This paper reports a decrease in the presence of the CD11c surface marker on mouse thymic dendritic cells (DC) as a result of host exposure to vanadium (here, in the form of vanadium pentoxide; V₂O₅) over a period of 4 weeks. All results were obtained using immunohistochemistry and flow cytometry. It is surmised that this decrease might induce a dysfunction, including possible negative selection of T-cells, which could increase the presence of autoreactive clones in the exposed host. Such an outcome could, in turn, increase the risk for development of autoimmune reactions in different organs specifically, and of autoimmune diseases in general in these V-exposed hosts.     

ＩＬ—６在Ｕ９３７细胞中特异、持续激活ＳＴＡＴ３

目的 探讨ＩＬ－６在Ｕ９３７细胞中产生生物学效应的信号转导基础。方法 检测ＩＬ－６对Ｕ９３７细胞生长与分化的影响。并采用凝胶阻滞电泳法,检测ＩＬ－６在Ｕ９３７细胞中激活核因子的情况。结果 ＩＬ－６可诱导Ｕ９３７细胞向巨噬细胞方向分化。分化的细胞酸性醋酸酯酶（ＡＮＡＥ）活性、硝基蓝四唑（ＮＢＴ）还原能力及ＣＤ５４表达增强,ＳＴＡＴ３可被ＩＬ－６显著激活,其活性呈一定的剂量与时间依赖性;而ＮＦ－ＩＬ６、ＮＦ－kB、ＡＰ－１及其它ＳＴＡＴ家族成员则无明显激活。结论 Ｕ９３７细胞的终分化可能是ＪＡＫ－ＳＴＡＴ３通路介导的。     

Induction of CD 14 antigen on the surface of U937 cells by an interleukin-6 autocrine mechanism after culture with formalin-killed Gram-negative bacteria

ABSTRACT: In order to better understand the regulation of CD 14 antigen on the surface of the monocyte-like cell line U937 in response to bacteria, the expression and regulation of CD 14 antigen on these cells when cultured with formalin-killed bacteria were determined using the monoclonal antibody MY-4 and analyzed by means of the indirect immunofluorescence method. CD14 expression was induced on the U937 cells after about 48 hours of culture with all of the formalin-killed Gram-negative bacteria used in this study but with none of the Gram-positive bacteria. Maximum expression was obtained after culture with formalin-killed Salmonella enteritidis strain 116–54. Various cytokines such as interleukin-1β, interleukin-2, interleukin-6, interferon-γ and tumor-necrosis factor-α were assayed in the culture supernatant of U937 cells cultured with or without formalin-killed Salmonella enteritidis 116–54 using an enzyme-immunoassay or radioimmunoassay system. The U937 cells were found to produce a large amount of interleukin-6 in response to formalin-killed Salmonella enteritidis 116–54. On the other hand, culture supernatant (referred to as conditioned medium) obtained from the U937 cells after 72 h of culture with formalin-killed Salmonella enteritidis 116–54 also induced strong expression of CD 14 antigen 48 to 72 h later, and this was blocked by the addition of anti-human interleukin-6 antibody. These findings suggest that the expression of CD 14 antigen on the surface of U937 cells cultured with formalin-killed Gram-negative bacteria is induced by interleukin-6 and can be explained on the basis of the autocrine mechanism of interleukin-6.     

Clq诱导Raji和U937细胞产生IL－1ra

Ｃｌｑ刺激Ｒａｊｉ和Ｕ９３７细胞２４ｈ，其上清中含ＩＬ－１抑制活性。抗人ｒＩＬ－１ｒａ抗血清能识别上清中一个约２２～２４ＫＤ的蛋白分子，并可中和其ＩＬ－１抑制活性，证实上清中的活性成份是ＩＬ－１ｒａ。Ｃｌｑ以特异、剂量依赖及可饱和的方式诱导这些细胞产生ＩＬ－１ｒａ，表明是Ｃｌｑ／ＣｌｑＲ系统介导了ＩＬ－１ｒａ的产生。资料提示：Ｃｌｑ／ＣｌｑＲ系统在炎症、免疫应答及细胞因子网络的调节中起重要作用。     

A monoclonal antibody, 3/22, to rabbit CD11c which induces homotypic T cell aggregation: evidence that ICAM-1 is a ligand for CD11c/CD18

The rabbit CD11c molecule has been characterized by use of a new monoclonal antibody, mAb 3/22. Expression of the p150,95 integrin (CD11c/CD18) has been shown by flow cytometry and immunohistochemistry to be restricted to monocytes, macrophages, dendritic cells and a small population of lymphocytes in peripheral blood. No expression on neutrophils could be demonstrated. Incubation of the newly derived CD8⁺ T cell line, BJ/873, with mAb 3/22 causes homotypic aggregation, which has been shown to be a cell surface event that is not dependent on intracellular signaling or on receptor cross-linking. Inhibition studies show that the ligands responsible for this aggregation are CD11c/CD 18 and ICAM-1, both of which are expressed on BJ/873. One other rabbit T cell line, K34, that also expresses p150,95 and ICAM-1, shows a similar aggregation response when stimulated with 3/22. Cell lines that express p150,95 but not ICAM-1 do not aggregate. These observations suggest that ICAM-1 is a ligand for activated p150,95.     

替米沙坦抑制U937细胞增殖及诱导凋亡

目的:探讨替米沙坦对U937细胞株的生长抑制及凋亡诱导作用。方法:分别以不同浓度的替米沙坦处理人类急性髓系白血病细胞U937;以CCK-8法检测不同浓度替米沙坦对U937细胞的生长抑制作用;以集落形成实验观察不同浓度替米沙坦对U937细胞集落形成能力的影响;以Annexin V-PI双染法及Hoechst 33342染色法检测不同浓度替米沙坦作用前后U937细胞凋亡程度的变化;以流式细胞术检测U937细胞表面抗原CD11b的阳性表达率,瑞氏染色后倒置显微镜进行细胞形态学观察,了解U937细胞的分化情况;以Western blot法检测不同浓度替米沙坦作用U937细胞后凋亡相关蛋白表达量的改变。结果:CCK-8实验结果证实替米沙坦呈时间和剂量依赖性抑制U937细胞的生长;集落形成实验显示低剂量替米沙坦可以完全抑制U937细胞的集落形成能力;Annexin V-PI双染法及Hoechst 33342法结果证实替米沙坦可以诱导U937细胞凋亡;细胞表面抗原流式检测术及瑞氏染色结果证实替米沙坦可以促进部分U937细胞分化;Western blot实验结果证实替米沙坦作用于U937细胞72 h后,促凋亡相关蛋白cleaved PARP及cleaved caspase-3蛋白的水平明显增高。结论:替米沙坦可以抑制细胞增殖以及诱导U937细胞部分分化,并通过caspase依赖的凋亡途径触发U937细胞凋亡。     

Renal‐infiltrating CD11c+ cells are pathogenic in murine lupus nephritis through promoting CD4+ T cell responses

Lupus nephritis (LN) is a major manifestation of systemic lupus erythematosus (SLE), causing morbidity and mortality in 40–60% of SLE patients. The pathogenic mechanisms of LN are not completely understood. Recent studies have demonstrated the presence of various immune cell populations in lupus nephritic kidneys of both SLE patients and lupus‐prone mice. These cells may play important pathogenic or regulatory roles in situ to promote or sustain LN. Here, using lupus‐prone mouse models, we showed the pathogenic role of a kidney‐infiltrating CD11c⁺ myeloid cell population in LN. These CD11c⁺ cells accumulated in the kidneys of lupus‐prone mice as LN progressed. Surface markers of this population suggest their dendritic cell identity and differentiation from lymphocyte antigen 6 complex (Ly6C)^low mature monocytes. The cytokine/chemokine profile of these renal‐infiltrating CD11c⁺ cells suggests their roles in promoting LN, which was confirmed further in a loss‐of‐function in‐vivo study by using an antibody‐drug conjugate (ADC) strategy targeting CX₃CR1, a chemokine receptor expressed highly on these CD11c⁺ cells. However, CX₃CR1 was dispensable for the homing of CD11c⁺ cells into lupus nephritic kidneys. Finally, we found that these CD11c⁺ cells co‐localized with infiltrating T cells in the kidney. Using an ex‐ vivo co‐culture system, we showed that renal‐infiltrating CD11c⁺ cells promoted the survival, proliferation and interferon‐γ production of renal‐infiltrating CD4⁺ T cells, suggesting a T cell‐dependent mechanism by which these CD11c⁺ cells promote LN. Together, our results identify a pathogenic kidney‐infiltrating CD11c⁺ cell population promoting LN progression, which could be a new therapeutic target for the treatment of LN.     

CD11c+ cells are significantly decreased in the duodenum, ileum and colon of dogs with inflammatory bowel disease

CD11c serves as a marker for human and murine dendritic cells (DCs) and cells expressing this marker have been shown to have similar morphological and functional characteristics in the canine immune system. The aim of this study was to quantify CD11c⁺ cells in the duodenum, ileum and colon of healthy dogs and dogs with inflammatory bowel disease (IBD). Endoscopic biopsies from the duodenum (n = 12 cases), ileum (n = 8 cases) and colon (n = 12 cases) were obtained from dogs diagnosed with IBD. Intestinal tissue from 10 healthy beagle dogs was used as control. Immunofluorescence microscopy was carried out using an anti-canine CD11c monoclonal antibody. Labelled cells were recorded as cells per 120,000 μm². The canine chronic enteropathy clinical activity index (CCECAI) was calculated for all dogs with IBD. In addition, sections from all dogs with IBD were evaluated according to the guidelines of the World Small Animal Veterinary Association Gastrointestinal Standardization Group. The number of CD11c⁺ cells in the duodenum, ileum and colon of dogs with IBD was significantly reduced compared with controls (P < 0.01, P < 0.01 and P < 0.05, respectively). There was a significant negative correlation between the number of CD11c⁺ cells in the colon of dogs with IBD and the CCECAI (P = 0.044, r² = −0.558). Chronic inflammation in canine IBD appears to involve an imbalance in the intestinal DC population. Future studies will determine whether reduced expression of CD11c could be a useful marker for the diagnosis and monitoring of canine IBD.     

Supplementation of CXCL12 (CXCL12) induces homing of CD11c+ dendritic cells to the spleen and enhances control of Plasmodium berghei malaria in BALB/c mice

In malaria, parasitaemia is controlled in the spleen, a multicomponent organ that undergoes changes in its cellular constituents to control the parasite. During this process, dendritic cells (DCs) orchestrate the positioning of effector cells in a timely manner for optimal parasite clearance. We have recently demonstrated that CXCL12 [stromal cell‐derived factor‐1 (CXCL12)] supplementation partially restores the ability to control parasitaemia in Plasmodium berghei‐infected mice. In the present study, we investigated the nature of the DCs involved by flow cytometry and immunohistochemistry of CD11c⁺ cells. Flow cytometry of bone marrow cells showed that infection with P. berghei did not alter the proportion of CD11c⁺ cells present in this haematopoietic compartment, while CXCL12 supplementation of naïve uninfected mice induced only minor increases in the population of CD11c⁺ cells. In the spleen, P. berghei infection alone resulted in an increase in CD11c⁺ cells as compared with naïve animals. Exogenously administered CXCL12 in the absence of infection resulted in a significant expansion of the splenic CD11c⁺ population, and this effect was even more pronounced in infected and supplemented mice. Immunohistochemistry revealed that CD11c⁺ cells infiltrated the perivascular areas and marginal zone of the spleen in infected animals treated with CXCL12, suggesting that this chemokine induces homing of CD11c⁺ dendritic cells to the splenic compartment. Our results show that small amounts of CXCL12 supplementation are effective in recruiting DCs to the spleens of both uninfected and infected mice, suggesting the participation of CXCL12 and CD11c⁺ cells in the establishment of an adequate environment in the spleen for malaria control.     

Depletion of CD11c+ cells in the CD11c.DTR model drives expansion of unique CD64+ Ly6C+ monocytes that are poised to release TNF‐α

Dendritic cells (DCs) play a vital role in innate and adaptive immunities. Inducible depletion of CD11c⁺ DCs engineered to express a high‐affinity diphtheria toxin receptor has been a powerful tool to dissect DC function in vivo. However, despite reports showing that loss of DCs induces transient monocytosis, the monocyte population that emerges and the potential impact of monocytes on studies of DC function have not been investigated. We found that depletion of CD11c⁺ cells from CD11c.DTR mice induced the expansion of a variant CD64⁺ Ly6C⁺ monocyte population in the spleen and blood that was distinct from conventional monocytes. Expansion of CD64⁺ Ly6C⁺ monocytes was independent of mobilization from the BM via CCR2 but required the cytokine, G‐CSF. Indeed, this population was also expanded upon exposure to exogenous G‐CSF in the absence of DC depletion. CD64⁺ Ly6C⁺ monocytes were characterized by upregulation of innate signaling apparatus despite the absence of inflammation, and an increased capacity to produce TNF‐α following LPS stimulation. Thus, depletion of CD11c⁺ cells induces expansion of a unique CD64⁺ Ly6C⁺ monocyte population poised to synthesize TNF‐α. This finding will require consideration in experiments using depletion strategies to test the role of CD11c⁺ DCs in immunity.     

Mycobacterium vaccae induces a population of pulmonary CD11c+ cells with regulatory potential in allergic mice

The hygiene hypothesis proposes that common, harmless microorganisms, present throughout our evolutionary history, have helped to develop immunoregulatory mechanisms that prevent inappropriate immune responses by the host. Using a mouse model of allergic pulmonary inflammation, we report that treatment with an ubiquitous saprophytic mycobacterium, Mycobacterium vaccae, significantly reduces allergic inflammation by decreasing type 2 responses such as eosinophilia and IL-4 expression. Rather than observing an increase in type-1 cytokine expression, we found elevated production of IL-10 in the lungs suggesting a role for regulatory T cells. Since induction of these cells may be dependent on APC, we investigated the effects of M. vaccae treatment on pulmonary CD11c+ cells. Increased levels of IL-10, TGF-beta and IFN-alpha mRNA were detected in CD11c+ cells from M. vaccae-treated allergic mice. We propose that M. vaccae-induced CD11c+ cells have a potential regulatory role at the site of inflammation through their secretion of immunomodulatory cytokines.     

Effect of cysteinyl leukotriene receptor antagonist on CD11b and CD23 expression in asthmatic children

BACKGROUND: Cysteinyl leukotrienes are potent pro-inflammatory mediators that contribute to the pathophysiologic features observed in allergic asthma. Inhibitors of leukotriene receptors represent novel therapy in asthma treatment. In addition to the protection from early asthmatic responses, these drugs have recently been shown to protect from late airway responses too. METHODS: We studied the effect of treatment with an oral antagonist of cysteinyl leukotriene receptors on the increased expression of the low-affinity IgE receptor, CD23, on B cells, and of its ligands, CD11b and CD11c, on CD4(+) T cells and monocytes in peripheral blood of patients with allergic asthma. In this uncontrolled open-label study, 14 children with allergic asthma received montelukast, a cysteinyl leukotrine receptor antagonist, for a period of 6 weeks after demonstrating forced expiratory volume in 1 s (FEV(1)) of less than 80% of the predicted value. Samples of peripheral heparinized blood and sera were obtained before and after therapy completion. Three-colour immunofluorescence analysis was performed, and expression of CD11b and CD11c on CD4(+) T lymphocytes and monocytes as well as the expression of CD21 and CD23 on B cells were determined (n=12). Peripheral blood eosinophil count, changes in FEV(1) and peak expiratory flow rate (PEFR), asthma exacerbations, and as-needed use of beta-agonist were also monitored. RESULTS: Montelukast improved FEV(1) and PEFR, and decreased peripheral eosinophil counts in all study patients. There was no significant change in the expression of CD21 and CD23 on B cells. The expression of CD11c on CD4(+) T cells and of both CD11b and CD11c on monocytes remained similar to the pretreatment expression. However, the percentage of CD11b(+)CD4(+) T lymphocytes significantly decreased after treatment with montelukast. This was accompanied by a significant decrease in the levels of total IgE. CONCLUSION: The capacity of 6-week montelukast therapy to reduce the percentage of CD11b CD4(+) T cells might be a mechanism leading to the immune response modulation on this T cell subset interaction with CD23-expressing B cells and subsequent down-regulation of IgE synthesis.