Effect of chloroquine on mast cell membranes

The effect of chloroquine (CQ) on phospholipid turnover andde novo synthesis in isolated rat mast cells (IRMC) was studied by determining the incorporation of³²P and¹⁴C-glycerol into IRMC phospholipids. Incubation of mast cells with chloroquine increased³²P incorporation into PI and PS whilst it decreased³²P incorporation into PC, PE and PA. In mast cells pretreated with CQ and subsequently stimulated with compound 48/80,³²P incorporation into PI, PS and PA fractions was enhanced, while it was decreased into PC and PE, in comparison to 48/80 stimulated IRMC. ¹⁴C-glycerol incorporation into total IRMC phospholipids was not significantly changed by CQ and compound 48/80 treatment and neither was any dose-dependent effect of CQ on individual phospholipids detected. Our results indicate that chloroquine, similarly to other cationic amphiphilic drugs, may alter membrane PL turnover without changingde novo synthesis of phospholipids.     

Effect of chloroquine on mast cell membranes

The effect of chloroquine (CQ) on phospholipid turnover andde novo synthesis in isolated rat mast cells (IRMC) was studied by determining the incorporation of³²P and¹⁴C-glycerol into IRMC phospholipids. Incubation of mast cells with chloroquine increased³²P incorporation into PI and PS whilst it decreased³²P incorporation into PC, PE and PA. In mast cells pretreated with CQ and subsequently stimulated with compound 48/80,³²P incorporation into PI, PS and PA fractions was enhanced, while it was decreased into PC and PE, in comparison to 48/80 stimulated IRMC.¹⁴C-glycerol incorporation into total IRMC phospholipids was not significantly changed by CQ and compound 48/80 treatment and neither was any dose-dependent effect of CQ on individual phospholipids detected. Our results indicate that chloroquine, similarly to other cationic amphiphilic drugs, may alter membrane PL turnover without changingde novo synthesis of phospholipids.     

羌活水提取物对肥大细胞活化的影响

目的:探讨羌活水提取物(EN)对肥大细胞活化的影响及其机制。方法:体外获取大鼠腹腔肥大细胞(RPMC),观察EN不同剂量对anti-DNP IgE诱导肥大细胞活化的影响,酶联法检测anti-DNP IgE介导的肥大细胞组胺释放,酶联免疫吸附测定法检测肥大细胞肿瘤坏死因子-α(TNF-α)和白细胞介素-6(IL-6)释放,免疫印迹检测丝氨酸/苏氨酸蛋白激酶(Akt)、核转录因子κB(NF-κB)p65蛋白的表达。结果:体外anti-DNP IgE明显促进RPMC组胺,肿瘤坏死因子-α(TNF-α)和白介素6(IL-6)的释放,诱导Akt磷酸化和NF-κB p65活化,羌活水提取物高(200μg/L)、中(100μg/L)浓度明显抑制肥大细胞组胺,TNF-α和IL-6的释放,并抑制Akt磷酸化和NF-κB p65活化。结论:羌活水提取物通过调节Akt,NF-κB p65信号通路抑制肥大细胞介导的过敏性炎症。     

Enhancement of mast cell differentiation in vitro by T cell factor(s)

Mast cell colonies were obtained when lymph node cells of horse serum-immunized Balb/c mice were cultured in a horse serum-containing medium on embryonic fibroblast monolayer. In order to characterized precursors of mast cells, mesenteric lymph node cells from the immunized mice were fractionated to obtain nonadherent cells, a B cell-depleted fraction and a T cell-depleted fraction; and each fraction was cultured on fibroblast monolayer. Mast cell colonies developed from nonadherent cells and from the B cell-depleted fraction but not from the T cell-depleted fraction. However, cultures of the same T cell-depleted fraction developed mast cell colonies if cell-free supernatant obtained from culture of horse serum-primed T cells was added. Soluble factors promoting mast cell growth were not obtained when the same T cells were incubated in horse serum-free medium. It appears that the majority of mast cell precursors in the lymph nodes are nonadherent cells and bear neither immunoglobulin nor Thy 1 antigen. The results also suggested that soluble factor(s) released from antigen-stimulated T cells enhanced the differentiation of the precursors to mature mast cells.     

Effect of human mast cell tryptase on human plasma proenzymes

The effect of human skin mast cell tryptase on human plasma proenzymes (prothrombin, coagulation factor XII, complement C1s, protein C and plasminogen) was investigated. Tryptase had no effect on these proenzymes, when incubated with them at 37 degrees C for up to 90 min, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the ability to hydrolyze specific peptide p-nitroanilide substrates. After prolonged treatment with tryptase, proenzymes could be fully activated with their specific activators. The results indicate that tryptase neither activates these plasma proenzymes nor inactivates the corresponding active enzymes. As a positive control, the tryptase preparation was also incubated with human fibrinogen and rat thymus histones. Prolonged treatment with tryptase increased the thrombin-induced clotting time of fibrinogen. Tryptase also efficiently hydrolyzed histone H1 from rat thymus. Histones H3/H2B and H2A were hydrolyzed less efficiently than H1, and no hydrolysis of histone H4 by tryptase was detected under the experimental conditions.     

Cytokine-induced human basophil/mast cell growth and differentiation in vitro

The growth and differentiation in vitro of rodent mast cells, a process dependent upon interleukin (IL)-3, has already been well established. Only recently, however, have the mechanisms underlying the development in vitro of human metachromatic cells (basophils and mast cells) begun to be delineated. Precursors of human metachromatic cells are found in bone marrow, peripheral blood, cord blood, fetal liver and are represented by some leukemic cell lines. These are dependent upon the presence of several cytokines or accessory cells for their proper growth and differentiation. IL-3 as well as granulocyte-macrophage/colony-stimulating factor (GM-CSF) appear to be the principal human metachromatic cell hemopoietic factors; contributory roles to metachromatic cell differentiation can also be shown for IL-5 and nerve growth factor. Stromal cell populations, including fibroblasts and epithelial cells, especially from allergic or inflamed tissue microenvironments, elaborate GM-CSF and possibly novel metachromatic cell differentiation factors. Questions remain regarding cell origins, specific hemopoietic factors and lineage inter-relationships for human mast cell subtypes and basophils. The intriguing possibility of mast cell-drived hemopoietic cytokines, which could perpetuate human allergic reactions, is currently under scrutiny. The relevance of existing data and future research in this area to diagnosis and therapy of a large group of human immune-inflammatory conditions is not to be underestimated.     

Investigation of mast cell differentiation in vivo by use of monoclonal antibodies

In the present study mast cell differentiation/maturation was studied in vivo after depletion of mature mast cells from the peritoneal cavity by injection of distilled water. The reconstituting cell population was characterized by use of different staining methods. Additionally, the monoclonal antibody (MAb) IWF F2, which recognizes a membrane antigen of rat mast cells, was used to follow up mast cell differentiation/maturation in the course of the experiment. The antigen expression was studied both by immunofluorescence of antigen-bearing cells and by MAb inhibition of compound 48/80-stimulated histamine release from mast cells. In the course of the experiment the amount of antigen-positive cells increased continuously from less than 5% to control level (1st and 22nd days, respectively). The expression of the membrane antigen detectable by the MAb precedes the appearance of cytochemically identifiable mast cells for several days. The mast cells mature morphologically and functionally as indicated by increasing size, histamine content and MAb inhibition of compound 48/80-stimulated histamine release. The results obtained suggest the MAb IWF F2 to be a useful methodical tool for additional characterization of mast cell differentiation/maturation processes.     

Acquisition and alteration of adhesion molecules during cultured human mast cell differentiation

BACKGROUND: Mature human mast cells express several types of adhesion molecules on their surface. Interactions between extracellular matrix (ECM) and adhesion molecules may be important for the migration and localization of mast cells and their precursors in tissues. Little is known about the regulation of adhesion molecules on mast cells during their differentiation. OBJECTIVES: To clarify the evolution of adhesion phenotype and function, we examined the expression of adhesion molecules during cultured human mast cell (CHMC) differentiation and tested adhesion of mature CHMCs to various ECM proteins. METHODS: CHMCs were obtained by culturing human cord blood-derived CD34(+) cells in the presence of stem cell factor and IL-6. Indirect immunofluorescence and flow cytometry was used to study cell surface expression of adhesion molecules and other markers. Mature CHMCs were tested for adhesion molecule function with immobilized matrix proteins. RESULTS: At 1 week of culture, cells expressed CD11a, CD18, CD29, CD49d, and CD49e. At 14 weeks of culture, more mature CHMCs expressed CD11b, CD11c, CD29, CD49b, CD49c, CD49d, CD49e, CD51, CD61, and CD54 and weakly expressed CD18 and CD11a. CD11c, CD51, and CD61 appeared de novo by 4 weeks of culture, whereas CD49b and CD49c appeared by 8 weeks. CD29 decreased at 4 weeks but returned to the identical levels of 1-week-old cells by 8 weeks. Compared with levels at week 1, the levels of CD11a, CD18, CD49d, and CD49e at 4 weeks and beyond decreased during culture. Expression of CD49a, CD49f, and alphad integrin was never detectable during CHMC differentiation. Fourteen-week-old CHMCs significantly adhered to the leucine-aspartic acid-valine-containing connecting segment 1 fragment of fibronectin, the 120-kd argine-glycine-aspartic acid-containing fragment of fibronectin, vitronectin, and laminin through specific integrins. CONCLUSION: Expression of integrins and CD54 is differentially regulated during CHMC differentiation, and mature CHMCs can adhere to many ECM proteins. These changes may facilitate emigration from the bone marrow into the circulation and ultimately contribute to the tissue homing and localization pattern seen with mature mast cells.     

Human mast cell proteases and mast cell heterogeneity

Mast cell neutral proteases are distinctive markers of the MC(T) and MC(TC) cells in humans. Measurements of tryptase levels in vivo serve as an overall indicator of mast cell activity. Further research is needed to evaluate the functional role of these proteases as well as each mast cell type in situations related to both health and disease.     

Rat cardiac mast cell maturation and differentiation following acute ventricular volume overload

Objective Cardiac mast cell numbers increase significantly within 12 h following the creation of an aortocaval (AV) fistula in rats and play a central role in mediating adverse left ventricular remodeling. We studied whether this increase was related to maturation of resident immature mast cells. Methods We measured percentages of immature and mature cardiac mast cells at 1, 2 and 7 days following AV-fistula or sham surgery and in non-surgical control rats using the alcian-blue safranin reaction. Results Relative to sham-operated and control rats, there was a significant shift from immature to a greater percentage of mature cardiac mast cells at 1 day and 2 days post-fistula that returned to a normal distribution by 7 days. Conclusions We conclude that the acute increase in mast cell density following volume overload is due to a paracrine response in the heart that stimulates the maturation and differentiation of resident immature cardiac mast cells. Received 10 June 2005; returned for revision 14 July 2005; accepted by A. Falus 29 March 2006     

木犀草素对肥大细胞脱颗粒影响及机制的研究

目的:探讨木犀草素(Luteolin)的抗I 型变态反应的作用机制。方法:通过建立DNP-BSA-IgE 激发致敏的大鼠RBL-2H3 细胞模型,分别采用MTT 法检测不同浓度(5、15、25 mol/ L)Luteolin 对RBL-2H3 肥大细胞活性的影响;ELISA 法检测不同浓度Luteolin 对RBL-2H3 细胞分泌 hexosa minidase( HEX)及细胞因子TNF 影响;Flou-4AM 荧光探针检测细胞内Ca2+浓度变化;Western blot 检测AKT,P-AKT 的表达。结果:成功建立致敏的细胞模型,低浓度的Luteolin 对RBL-2H3 细胞活性无明显影响;不同浓度Luteolin 刺激RBL-2H3 细胞后,对 HEX 和TNF鄄琢的释放抑制作用呈线性相关,且细胞内Ca2+ 明显减少;Western blot 结果显示随着Luteolin 浓度的增加AKT 磷酸化水平明显下降。结论:Luteolin 呈剂量依赖性抑制RBL鄄2H3 肥大细胞脱颗粒,且通过调节细胞内Ca2+浓度与AKT 活性参与其中。     

Effect of lipopolysaccharide on mouse mast cell induction by a splenic cell culture system.

We have previously reported a method of mast cell induction by long-term culture of mouse spleen cells without using exogenous mast cell growth factor (Z.-Q. Hu, T. Yoshida, and T. Shimamura, J. Immunol. Methods 149:173, 1992). Supernatants recovered from the long-term cultures contain endogenous interleukin 3 and soluble stem cell factor. These were assessed by the capacity of the recovered supernatants to foster the growth of a mast cell growth factor-dependent cell line and by neutralizing antibodies. Besides the soluble factors, cell-to-cell contacts mediated by membrane stem cell factor on splenic stromal cells and c-Kit receptors on mast cells also affect mast cell induction. Different lots of fetal calf serum (FCS) were examined to determine a possible trigger for cytokine production. FCS can be divided into mast cell-inducible and noninducible sera by this process. However, not all FCS lots contain mast cell growth factor. The mast cell-inducible lots contain lipopolysaccharide (LPS) confirmed by a Limulus assay. Polymyxin B can neutralize the mast cell induction activity. Non-mast cell-inducible FCS can be converted to inducible FCS by adding exogenous LPS. The results indicate that LPS as a trigger of cytokine production is responsible for mast cell induction.