Antagonism of the final common pathway of mast cell histamine secretion by arylalkylamines

The antagonism of histamine secretion from rat peritoneal mast cells by a range of drugs (e.g., antihistamines, local anesthetics, tranquilizers, antidepressants, adrenergics and antiadrenergics) and arylalkylamines was studied. Simple arylalkylamines, those without quaternary ammonium, phenolic or anionic groups, uniformly inhibited histamine secretion induced via three major secretory pathways of mast cells (compound 48/80, A23187 and concanavalin A). That is, each simple arylalkylamine nonselectively inhibited all three secretagogues at about the same concentration (avg. range = 2.2). This nonselective inhibition was rapid in onset (seconds), readily reversible by washing and not related to a decrease in ATP. It is likely that these agents act nonspecifically at a step in the final common pathway in the cell membrane, possibly to inhibit fusion of the secretory granules with the plasma membrane. Other, more complex arylalkylamines were selective in their antagonism of histamine secretion. Quaternary arylalkylammonium compounds selectively antagonized polyamine secretagogues (48/80 and polymyxin B), whereas arylalkylamines, with other hydrophilic groups, selectively inhibited one or another of the secretory pathways. These complex arylalkylamines must act before the final common pathway because of their selective inhibition. At higher concentrations most of the arylalkylamines caused disruption of the mast cells and liberated histamine.     

iNKT cells require TSC1 for terminal maturation and effector lineage fate decisions

Terminal maturation of invariant NKT (iNKT) cells from stage 2 (CD44⁺NK1.1^–) to stage 3 (CD44⁺NK1.1⁺) is accompanied by a functional acquisition of a predominant IFN-γ–producing (iNKT-1) phenotype; however, some cells develop into IL-17–producing iNKT (iNKT-17) cells. iNKT-17 cells are rare and restricted to a CD44⁺NK1.1^– lineage. It is unclear how iNKT terminal maturation is regulated and what factors mediate the predominance of iNKT-1 compared with iNKT-17. The tumor suppressor tuberous sclerosis 1 (TSC1) is an important negative regulator of mTOR signaling, which regulates T cell differentiation, function, and trafficking. Here, we determined that mice lacking TSC1 exhibit a developmental block of iNKT differentiation at stage 2 and skew from a predominantly iNKT-1 population toward a predominantly iNKT-17 population, leading to enhanced airway hypersensitivity. Evaluation of purified iNKT cells revealed that TSC1 promotes T-bet, which regulates iNKT maturation, but downregulates ICOS expression in iNKT cells by inhibiting mTOR complex 1 (mTORC1). Furthermore, mice lacking T-bet exhibited both a terminal maturation defect of iNKT cells and a predominance of iNKT-17 cells, and increased ICOS expression was required for the predominance of iNKT-17 cells in the population of TSC1-deficient iNKT cells. Our data indicate that TSC1-dependent control of mTORC1 is crucial for terminal iNKT maturation and effector lineage decisions, resulting in the predominance of iNKT-1 cells.     

Synergy between the pre-T cell receptor and Notch: cementing the alphabeta lineage choice

Notch1 signaling suppresses B cell development and promotes T lineage commitment in thymus-seeding hematopoietic progenitors. Notch1 is also activated in early T cell progenitors, but the functions of these later Notch signals have not been clearly defined. Recent studies reveal that Notch signaling is not essential for pre-T cell receptor (TCR) expression or gammadelta lineage choice. Rather, pre-TCR signaling enhances progenitor competitiveness for limiting Notch ligands, leading to preferential expansion of TCRbeta-bearing progenitors.     

Differential synergy of Notch and T cell receptor signaling determines alphabeta versus gammadelta lineage fate

Thymic precursors expressing the pre-T cell receptor (TCR), the gammadeltaTCR, or the alphabetaTCR can all enter the CD4+ 8+ alphabeta lineage, albeit with different efficacy. Here it is shown that proliferation and differentiation of precursors with the different TCRs into alphabeta lineage cells require Notch signaling at the DN3 stage of thymic development. At the DN4 stage, Notch signaling still significantly contributes to the generation of alphabeta T cells. In particular, in alphabeta lineage commitment, the pre-TCR synergizes more efficiently with Notch signals than the other two TCRs, whereas gammadeltaTCR-expressing cells can survive and expand in the absence of Notch signals, even though Notch signaling enhances their proliferation. These observations suggest a new model of alphabeta versus gammadelta lineage choice in which lineage fate is determined by the extent of synergy between TCR and Notch signaling and in which the evolutionarily recent advent of the cell-autonomously signaling pre-TCR increased the efficacy of alphabeta T cell generation.     

Rearrangement and selection of VH11 in the Ly-1 B cell lineage

One of the predominant VH genes utilized to encode the anti-BrMRBC specificity is a member of the small VH11 family rearranged to JH1. Using the polymerase chain reaction (PCR) we have determined that the frequency of B cells with a VH11 rearrangement is 10-20 times higher in Ly-1 B than in Ly-1- "conventional" B cells regardless of location (spleen or peritoneal cavity). Conventional B cells rearrange this gene at comparable levels in pre-B cells and in mature B cells utilizing all JH gene segments. In contrast, the increased levels of VH11 rearrangement in Ly-1 B are restricted to JH1 (and some JH2) and therefore appear to be the result of selection. Furthermore, most peritoneal Ly-1 B cells with VH11 rearrangements fall in a fraction stained by anti-BrMRBC antibody, likely bearing multivalent natural (likely self) antigen constitutively bound to their surface Ig receptors. Thus, we suggest that autoantigens are largely responsible for the accumulation of autoantibody specificities in the Ly-1 B cell lineage with time, whereas they do not exert this effect in the conventional B cells.     

An essential role for RasGRP1 in mast cell function and IgE-mediated allergic response

Cross-linking of the FcepsilonRI activates the phosphatidyl inositol 3 kinase (PI3K) and mitogen-activated protein kinase pathways. Previous studies demonstrate that Ras guanyl nucleotide-releasing protein (RasGRP)1 is essential in T cell receptor-mediated Ras-Erk activation. Here, we report that RasGRP1 plays an important role in FcepsilonRI-mediated PI3K activation and mast cell function. RasGRP1-deficient mice failed to mount anaphylactic allergic reactions. RasGRP1-/- mast cells had markedly reduced degranulation and cytokine production. Although FcepsilonRI-mediated Erk activation was normal, PI3K activation was diminished. Consequently, activation of Akt, PIP3-dependent kinase, and protein kinase C delta was defective. Expression of a constitutively active form of N-Ras could rescue the degranulation defect and Akt activation. We further demonstrated that RasGRP1-/- mast cells were defective in granule translocation, microtubule formation, and RhoA activation. Our results identified RasGRP1 as an essential regulator of mast cell function.     

Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1

Expulsion of gastrointestinal nematodes is associated with pronounced mucosal mast cell (MMC) hyperplasia, differentiation, and activation, accompanied by the systemic release of MMC granule chymases (chymotrypsin-like serine proteases). The beta-chymase mouse mast cell protease-1 (mMCP-1) is expressed predominantly by intraepithelial MMCs, and levels in the bloodstream and intestinal lumen are maximal at the time of worm expulsion in parasitized mice. To address the in vivo functions of MMC-specific beta-chymases, we have generated transgenic mice that lack the mMCP-1 gene. They were backcrossed onto a congenic BALB/c background to investigate the response to nematode infection. The deletion of the mMCP-1 gene is associated with significantly delayed expulsion of Trichinella spiralis and increased deposition of muscle larvae in BALB/c mice despite the presence of normal and sometimes increased numbers of MMCs. Neither worm fecundity nor worm burdens were altered in Nippostrongylus-infected mMCP-1(-/)- BALB/c mice. These data demonstrate, for the first time, that the ablation of an MMC-derived effector molecule compromises the expulsion process.     

肥大细胞的研究进展

肥大细胞(MC)是系统发生学的一种成熟细胞。在哺乳动物,MC分布于除骨、软骨和眼球角膜的结缔组织,并以相当一致方式围绕血管、淋巴管、和神经末梢分布。MC是一种成活时间长的多功能分泌细胞,胞浆内有大量由蛋白多糖基质、肝素组成的高电子密度颗粒。它是哺乳动物组织内组胺的主要储藏地。本文就MC的表型、测定方法、介质等方面的研究现状作一简要介绍。     

Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus

We have recently reported that Notch 1, a member of the Notch multigene family, is essential for the development of murine T cells. Using a mouse model in which Notch 1 is inactivated in bone marrow (BM) precursors we have shown that B cells instead of T cells are found in the thymus of BM chimeras. However, it is not clear whether these B cells develop by default from a common lymphoid precursor due to the absence of Notch 1 signaling, or whether they arise as a result of perturbed migration of BM-derived B cells and/or altered homeostasis of normal resident thymic B cells.In this report we show that Notch 1-deficient thymic B cells resemble BM B cells in phenotype and turnover kinetics and are located predominantly in the medulla and corticomedullary junction. Peripheral blood lymphocyte analysis shows no evidence of recirculating Notch1(-/)- BM B cells. Furthermore, lack of T cell development is not due to a failure of Notch1(-/)- precursors to home to the thymus, as even after intrathymic reconstitution with BM cells, B cells instead of T cells develop from Notch 1-deficient precursors. Taken together, these results provide evidence for de novo ectopic B cell development in the thymus, and support the hypothesis that in the absence of Notch 1 common lymphoid precursors adopt the default cell fate and develop into B cells instead.     

Delta-like 4 is the essential, nonredundant ligand for Notch1 during thymic T cell lineage commitment

Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor–mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1- and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifically inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the first steps of intrathymic T cell development.     

精神科医生选择抗抑郁药物时最先考虑的影响因素

目的:探讨精神科医生选用抗抑郁剂对抑郁症患者进行干预时优先考虑的因素。方法:调查于2004-01/10在广州市脑科医院芳村、荔湾两个门诊部完成。就诊的患者主要来自广州地区,共1496例,平均年龄45岁,女性占多数(n=923,61.7%)。调查问卷由作者自行编制,含40个条目,罗列了可能影响医生选择用药的相关因素。问卷上不记录患者姓名,但记录患者性别、年龄等。医生在问卷上注明选用的药物,每个条目只需回答是或否。医生在开药之后立即完成问卷。本调查不涉及患者隐私,基本不与患者的利益冲突,故未签署知情同意书。结果:1496例患者全部进入结果分析。最常见的影响因素依次为患者特异性的临床症状或症状谱(54.3%)、避开特定的副作用(48.9%)以及并发症的存在(46.4%)等。而停药综合征(1.2%)及药物之间的相互作用(6.3%)则很少影响医生的用药选择。结论:抑郁症患者的并发症(如合并焦虑或焦虑症)、药物副作用以及抗抑郁剂对不同症状的作用特征为精神科医生选择抗抑郁剂时的主要考虑因素。     

精神科医生选择抗抑郁药物时最先考虑的影响因素

目的：探讨精神科医生选用抗抑郁剂对抑郁症患者进行干预时优先考虑的因素。方法：调查于2004-01／10在广州市脑科医院芳村、荔湾两个门诊部完成。就诊的患者主要来自广州地区，共1496例，平均年龄45岁，女性占多数（n=923，61．7％）。调查问卷由作者自行编制，含40个条目，罗列了可能影响医生选择用药的相关因素。问卷上不记录患者姓名，但记录患者性别、年龄等。医生在问卷上注明选用的药物，每个条目只需回答是或否。医生在开药之后立即完成问卷。本调查不涉及患者隐私，基本不与患者的利益冲突，故未签署知情同意书。结果：1496例患者全部进入结果分析。最常见的影响因素依次为患者特异性的临床症状或症状谱（54.3％）、避开特定的副作用（48．9％）以及并发症的存在（46．4％）等。而停药综合征（1．2％）及药物之间的相互作用（6．3％）则很少影响医生的用药选择。结论：抑郁症患者的并发症（如合并焦虑或焦虑症）、药物副作用以及抗抑郁刺对不同症状的作用特征为精神科医生选择抗抑郁剂时的主要考虑因素。     

Cloning and cellular localization of the rat mast cell 78-kDa protein phosphorylated in response to the mast cell "stabilizer" cromolyn

Disodium cromoglycate (cromolyn) inhibits mast cell secretion, but its mechanism has not been elucidated. One possibility is the phosphorylation of a 78-kDa mast cell protein, two fragments of which are homologous to moesin, a member of the ezrin, radixin, moesin family. These proteins appear to be involved in signal transduction by regulating functional associations between the cell surface and the cytoskeleton. Moesin cDNA was cloned from rat basophil leukemia cells, which are similar to mucosal mast cells, and polyclonal antiserum was prepared against recombinant moesin expressed in Escherichia coli. Moesin phosphorylated in mast cells treated with cromolyn shifted from the soluble to the precipitable fraction and associated with Sepharose-linked beta-actin. Recombinant moesin also associated with Sepharose-linked beta-actin, and so did purified RBL moesin, but only if the latter was first denatured. Moesin thus appears to have actin binding sites that are not exposed under normal conditions but may become available by in vivo phosphorylation or by denaturation. Immunocytochemistry using confocal microscopy showed moesin to be primarily localized on the inner aspect of the plasma membrane and around secretory granules. Double immunocytochemistry for moesin and actin colocalized them in most areas. Ultracryoimmunoelectron microscopy to preserve the antigenicity of moesin identified the protein close to the plasma and secretory granule membranes. Cromolyn appeared to induce clustering of moesin around secretory granules. It is hypothesized that conformational changes of moesin, regulated by phosphorylation/dephosphorylation, may lead to positional rearrangements with respect to the membrane/cytoskeleton that could possibly regulate mast cell secretion.     

Immunofluorescent staining for mast cells in idiopathic pulmonary fibrosis: quantification and evidence for extracellular release of mast cell tryptase.

In many diseases, retrospective analysis for determining the presence of mast cells has been difficult because of their loss of metachromatic staining properties once tissue has undergone formalin fixation. We quantified mast cells in peribronchiolar tissue of idiopathic pulmonary fibrosis (IPF) and in normal human lung by using rabbit antiserum to human mast cell tryptase. In lung biopsy specimens from 15 patients with IPF, the mean number of mast cells per high-power field in connective tissue directly adjacent to the lumen of small airways (0.5 to 2 mm in diameter) and other fibrotic foci was 29.9 +/- 10.8 in comparison with 13.7 +/- 3.5 in 16 normal controls (P < 0.001). In addition, mast cells in cases of IPF had an altered appearance--irregularity of the plasma membrane and release of extracellular tryptase. We conclude that the number of mast cells is increased in IPF and that the altered appearance of the mast cells suggests that they are activated and undergoing degranulation.