The role of Ras guanine nucleotide releasing protein 4 in Fc epsilonRI-mediated signaling, mast cell function, and T cell development

The RasGRP (Ras guanine nucleotide-releasing protein) family proteins are guanine nucleotide exchange factors that activate Ras GTPases, ultimately leading to MAPK activation and many cellular processes. The RasGRP family has four members. Published studies demonstrate that RasGRP1, RasGRP2, and RasGRP3 play critical roles in T cells, platelets, and B cells, respectively. RasGRP4 is highly expressed in mast cells. Although previous data suggest that it is important in mast cell development and function, the role of RasGRP4 in mast cells and allergic responses has not been clearly demonstrated. In this study, we generated RasGRP4(-/-) mice to examine the function of RasGRP4. Analyses of these mice showed that mast cells were able to develop normally in vivo and in vitro. Despite high levels of RasGRP4 expression in mast cells, RasGRP4 deficiency led to only a modest reduction in FcεRI-mediated degranulation and cytokine production. Interestingly, mast cells deficient in both RasGRP1 and RasGRP4 had a much more severe block in FcεRI-mediated signaling and mast cell function. We also made the unexpected finding that RasGRP4 functions during thymocyte development. Our data suggest that after the engagement of immunoreceptors, immune cells likely employ multiple members of the RasGRP family to transduce critical signals.     

植物抗坏血酸的生物合成、转运及其生物学功能

抗坏血酸是高等植物中普遍存在的小分子物质,在植物细胞对氧化胁迫的抵抗、细胞分裂和伸长中发挥着重要作用。它在线粒体中合成,然后被转运到其他细胞区间以发挥生理作用。在不同生物膜上可能存在不同的ASC转运体,这些转运体的存在可使ASC及其不同氧化还原态形式根据细胞生理状况的需要而在不同细胞区间转运。ASC的重要生理功能是与其氧化还原状态以及生物合成、代谢、再生和转运的相关酶类活性的变化密切相关的。本文简要综述了近年来人们在植物抗坏血酸生物合成、代谢、转运、逆境响应与植物生长发育中的研究进展。     

Mast cell granule composition and tissue location--a close correlation

This review provides a survey on mast cell heterogeneity, with aspects differing in humans and rodents or which are subject of conflicting evidence being discussed in greater detail. Mast cell subsets have been first defined in rats by their fixation and dye-binding properties, and detailed studies in humans and pigs reveal very similar observations. The dye-binding properties of rat mast cell subsets are causally related to the absence or presence of heparin in their granules. In humans, this relation has not been shown. Rodent mast cell subsets store different chymase-isoforms. In contrast, just a single chymase has been defined in humans, and mast cells are classified by the presence or relative absence of this chymase. Different investigators find quite different proportions of chymase-positive to chymase-negative mast cells. Tryptase(s) are found in most or every human mast cell, but in rodents, they have hitherto been essentially localised to mast cells in connective tissues. Human mast cell subsets may also be defined by their expression of receptors such as C5aR and possibly the beta-chemokine receptor CCR3; the CCR3 expression seems to be related to the human mast cell chymase expression. Ultrastructural studies are helpful to distinguish human mast cell subsets, and allow to distinguish between chronic and acute activation. The phenotypical characteristics may change in association with inflammation or other disease processes. Studies in humans and pigs show changed dye-binding and fixation properties of the granules. Experimental rodent infection models reveal similar changes of chymase isoform expression. Human lung mast cells have been reported to strongly upregulate their chymase content in pulmonary vascular disease. This line of evidence can explain some inconsistent information on mast cell heterogeneity and may help to understand the physiological role of mast cells.     

Bovine mast cells: distribution, density, heterogeneity, and influence of fixation techniques

Mast cells can be distinguished according to various characteristics: rodent mast cells have been subtyped by histochemical criteria, whereas canine and human mast cells have been classified according to their proteases. Comparisons of mast cells from different species have therefore resulted in contradictory and confusing opinions on mast cell heterogeneity. Thus, it is essential to obtain species-specific data on mast cell density and heterogeneity. The present study was carried out to determine the physiological distribution of mast cell numbers and types in bovines according to tissue location, staining, and fixation methods. Samples were fixed in formalin or Carnoy’s fluid. The average number of mast cells was determined by using a metachromatic staining method. Protease content of mast cells was examined with a double-enzyme-immunohistochemical staining technique. Three mast cell subtypes were distinguished: T-, TC-, and C-mast cells. The T-mast cell was the predominant subtype in nearly all investigated organs and tissue locations. Only tryptase-positive mast cells could be demonstrated in bovine skin and uterus. No chymase activity was found in these organs, regardless of the fixation type. A larger number of mast cells was observed after fixation in Carnoy’s fluid. The three different mast cell subtypes were only demonstrated in formalin-fixed tissue; chymase-positive mast cells were not found after fixation in Carnoy’s fluid. Increasing experimental data suggest that mast cell subtypes have different functions in promoting and modulating inflammation and in remodeling the extracellular matrix. Since mast cell tryptase and chymase have different functional properties, these results may clarify the different reaction patterns observed in various organs and species.     

Mast cell heterogeneity

Increasing evidence for the existence of inter- and intra-species mast cell heterogeneity has expanded the potential biological role of this cell. Early studies suggesting that mast cells at mucosal sites differ morphologically and histochemically from connective tissue mast cells have been confirmed using isolated intestinal mucosal mast cells in the rat and more recently in man. These studies also established that mucosal mast cells are functionally distinct from connective tissue mast cells. Thus, mucosal and connective tissue mast cells differ in their responsiveness to a variety of mast cell secretagogues and antiallergic agents. Speculation about the therapeutic use of antiallergic drugs in disorders involving intestinal mast cells cannot, therefore, be based on extrapolation from studies of their effects on mast cells from other sites. Regulatory mechanisms for mast cell secretion may also be heterogeneous since mucosal mast cells differ from connective tissue mast cells in their response to a variety of physiologically occurring regulatory peptides. The development of techniques to purify isolated mast cell subpopulations will facilitate future analysis of the biochemical basis of the functional heterogeneity of mast cells.     

Developmental roles of platelet-derived growth factors

Platelet-derived growth factor (PDGF) was originally identified in platelets and in serum as a mitogen for fibroblasts, smooth muscle cells (SMC) and glia cells in culture. PDGF has since expanded to a family of dimers of at least four gene products, whose biological actions are mediated through two receptor tyrosine kinases, PDGFRs. The present review summarizes and discusses the biological functions of PDGFs and PDGFRs in developmental processes, mainly as revealed through genetic analysis in mice. Such studies have demonstrated multiple critical roles of PDGFs and PDGFRs in embryonic and postnatal development. PDGFs seem to act upon specific populations of progenitor cells that give rise to several different cell types with distinct functions in a variety of developmental processes. Analogies are seen between the cell functions and the developmental processes controlled by PDGFs. This suggests that ancestral PDGF and PDGFR expression patterns and functions may have been iterated in related sets of morphogenetic processes in the course of evolution.     

Cooperation between mast cell carboxypeptidase A and the chymase mouse mast cell protease 4 in the formation and degradation of angiotensin II

The octapeptide angiotensin II (Ang II) exerts a wide range of effects on the cardiovascular system but has also been implicated in the regulation of cell proliferation, fibrosis, and apoptosis. Ang II is formed by cleavage of Ang I by angiotensin-converting enzyme, but there is also evidence for non-angiotensin-converting enzyme-dependent conversion of Ang I to Ang II. Here we address the role of mast cell proteases in Ang II production by using two different mouse strains lacking mast cell heparin or mouse mast cell protease 4 (mMCP-4), the chymase that may be the functional homologue to human chymase. Ang I was added to ex vivo cultures of peritoneal cells, and the generation of Ang II and other metabolites was analyzed. Activation of mast cells resulted in marked increases in both the formation and subsequent degradation of Ang II, and both of these processes were strongly reduced in heparin-deficient peritoneal cells. In the mMCP-4(-/-) cell cultures no reduction in the rate of Ang II generation was seen, but the formation of Ang-(5-10) was completely abrogated. Addition of a carboxypeptidase A (CPA) inhibitor to wild type cells caused complete inhibition of the formation of Ang-(1-9) and Ang-(1-7) but did not inhibit Ang II formation. However, when the CPA inhibitor was added to the mMCP-4(-/-) cultures, essentially complete inhibition of Ang II formation was obtained. Taken together, the results of this study indicate that mast cell chymase and CPA have key roles in both the generation and degradation of Ang II.     

Prostaglandin D2 receptors DP and CRTH2 in the pathogenesis of asthma

Prostaglandin D2 (PGD2) is a major prostanoid produced mainly by mast cells in allergic diseases, including bronchial asthma. However, its role in the pathogenesis of asthma remains unclear. PGD2-induced vasodilatation and increased permeability are well-known classical effects that may facilitate transendothelial migration of inflammatory cells, such as eosinophils, mast cells, lymphocytes, and monocytes in allergic inflammation. These effects are initiated via a PGD2 receptor, D prostanoid receptor (DP), and are referred to as DP-mediated vasodilation-extravasation. Recently, novel functions of DP have been identified. Furthermore, a novel and different receptor of PGD2, CRTH2, has been discovered. To date, DP and CRTH2 have been shown to be major PGD(2)-related receptors that have pivotal roles in mediating allergic diseases by effects such as directly regulating the migration of inflammatory cells and controlling the production of cytokines and lipid mediators. Available evidence suggests that CRTH2 and DP may collaborate in allergic inflammation. This review focuses on the novel roles of DP and CRTH2 in the initiation and maintenance of allergy.     

Mast cells in tumor growth: Angiogenesis,tissue remodelling and immune-modulation

There is a growing acceptance that tumor-infiltrating myeloid cells play an active role in tumor growth and mast cells are one of the earliest cell types to infiltrate developing tumors. Mast cells accumulate at the boundary between healthy tissues and malignancies and are often found in close association with blood vessels within the tumor microenvironment. They express many pro-angiogenic compounds, and may play an early role in angiogenesis within developing tumors. Mast cells also remodel extracellular matrix during wound healing, and this function is subverted in tumor growth, promoting tumor spread and metastasis. In addition, mast cells modulate immune responses by dampening immune rejection or directing immune cell recruitment, depending on local stimuli. In this review, we focus on key roles for mast cells in angiogenesis, tissue remodelling and immune modulation and highlight recent findings on the integral role that mast cells play in tumor growth. New findings suggest that mast cells may serve as a novel therapeutic target for cancer treatment and that inhibiting mast cell function may lead to tumor regression.     

Changes in number of serotonin-containing cells and serotonin levels in the intestinal mucosa of rats with colitis induced by dextran sodium sulfate

The role of serotonin in the pathogenesis of inflammatory bowel disease has not been fully studied. We examined the changes in the serotonin level and the density of serotonin-containing enterochromaffin (EC) and mast cells in the intestinal mucosa of dextran sodium sulfate (DSS)-induced colitis in rats. Rats were treated with 1.5% DSS for 1 month. Serotonin levels were biochemically measured and the density of epithelial EC cells and mucosal mast cells was quantified by serotonin immunohistochemistry. DSS caused malnutrition due to chronic diarrhea. Infiltrated inflammatory cells were microscopically observed in the colonic wall with intact epithelium. The serotonin content in the mucosa/submucosa tissue was increased in the proximal and distal colon in DSS-treated rats, compared to that in control rats. The density of EC cells in the epithelium also increased in the proximal and distal colon in DSS-treated rats. In contrast, the density of mast cells in the lamina propria dramatically increased in the distal, but not in the proximal colon in DSS-treated rats. This discrepancy implies the serotonin released from EC cells and from mast cells may play different roles in the pathogenesis of DSS-induced colitis. Accepted: 30 July 1999     

Carnosine attenuates mast cell degranulation and histamine release induced by oxygen-glucose deprivation

Carnosine (beta-alanyl-histidine) is a naturally occurring dipeptide that has been characterized as a putative hydrophilic antioxidant. The protective function of carnosine has been demonstrated in neuronal cells under ischemic injury. The purpose of this study was to investigate the effects of carnosine on oxygen-glucose deprivation (OGD)-induced degranulation and histamine release from mast cells. Cultured mast cells were exposed to OGD for 4 h, and then the degranulation was observed immediately by microscopy. Histamine release was analyzed by high-performance liquid chromatography (HPLC). OGD caused degranulation of mast cells, and increased histamine and lactate dehydrogenase (LDH) release. Carnosine (at a concentration of 5 mM) alone did not produce any appreciable effect on degranulation, histamine, and LDH release from mast cells under normal condition, but significantly inhibited the degranulation, histamine, and LDH release of mast cells induced by OGD. These results indicate that carnosine can protect mast cells from degranulation and histamine release and it may be an endogenous mast cell stabilizer in the pathological processes induced by ischemia.     

Mast cells and hemangioma

Hemangioma is a primary tumor of the microvasculature in which angiogenesis is initially excessive, followed by spontaneous regression of the newly formed vessels, with the cellular parenchyma gradually being replaced with fibrofatty tissue. Mast cells, which are highly heterogenous in terms of their morphology, function, and metabolic products, have been implicated in the pathophysiology of hemangioma. Csaba stain shows that mast cells are predominantly of the biogenic amine phenotype throughout the development of hemangioma. The predominance of this phenotype remains unaltered following successful steroid therapy, although their number increases fourfold. Mast cells, all of which stain positive for tryptase, and those that stain positive for chymase as well, have been identified in hemangioma biopsy specimens throughout the three developmental phases. The total number of mast cells is highest during the involuting phase, less in the involuted phase, and least in the proliferative phase. The proportion of mast cells that contain both tryptase and chymase decreases from the proliferative through involuting to the involuted phase. This decreasing proportion of mast cells that contain both tryptase and chymase with ongoing involution parallels that of progressive deposition of the extracellular matrix as indicated by increasing fibrosis and fatty deposition. The short-chain type VIII collagen, thought to play a key role in angiogenesis, has been detected throughout the developmental phases of hemangioma. It has been postulated that this collagen, which is produced early in new vessel development, provides a substratum to facilitate the migration of endothelial cells. It may also facilitate the deposition of other extracellular constituents and influence cell movement and the maintenance of cell phenotypes. The intracellular localization of type VIII collagen in mast cells only in the early proliferative phase suggests that there is an active synthesis by mast cells during this phase. The increasing extracellular localization during hemangioma development may be caused by an increased secretion of protein from intracellular stores. The increased number of mast cells during the involuting phase indicates that these cells may play a role in the regression of hemangioma. This is in contrast to the large body of evidence showing the proangiogenic role of mast cells. The proportion of proliferating mast cells decreases, whereas the proportion of mast cells positive for clusterin/apolipoprotein J increases with ongoing involution of hemangioma. Clusterin/apolipoprotein J expression has been considered as a prominent marker of apoptotic cell loss. The presence of clusterin/apolipoprotein J granules both in the adjacent endothelial cells and in capillary lumens suggests that mast cells may be secreting this apoptotic modulator to promote the regression of hemangioma. Certain effectors produced by mast cells may participate in the development of hemangioma. It has been proposed that one of the functions of mast cells is to release factors leading to the regression of hemangioma. The evidence suggests that although mast cells may have a function in the endothelial proliferation in hemangioma, they also play a crucial role in the regression of this tumor. However, the roles of mast cells in the life cycle of hemangioma are likely to be complex and may involve stimulators of angiogenesis in the proliferative phase but inhibitors in later phases.     

Hyaluronan: a multifunctional, megaDalton, stealth molecule

Hyaluronan has been implicated in biological processes such as cell adhesion, migration and proliferation. Traditionally, it was thought to be associated with the extracellular matrix, but, hyaluronan may also have unimagined roles inside the cell. Investigation of hyaluronan synthesis and degradation, the identification of new receptors and binding proteins, and the elucidation of hyaluronan-dependent signaling pathways are providing novel insights into the true biological functions of this fascinating molecule.     

In activated mast cells, IL-1 up-regulates the production of several Th2-related cytokines including IL-9

Mast cells can play detrimental roles in the pathophysiology and mortality observed in anaphylaxis and other Th2-dominated allergic diseases. In contrast, these cells contribute to protective host defense mechanisms against parasitic worm infections. After IgE/Ag activation, mast cells can produce multiple cytokines that may enhance allergic inflammations, while a similar panel of Th2-related cytokines may support immunological strategies against parasites. Here we report that in primary mouse bone marrow-derived mast cells activated by ionomycin or IgE/Ag, the proinflammatory mediator IL-1 (alpha or beta) up-regulated production of IL-3, IL-5, IL-6, and IL-9 as well as TNF, i.e., cytokines implicated in many inflammatory processes including those associated with allergies and helminthic infections. IL-1 did not induce significant cytokine release in the absence of ionomycin or IgE/Ag, suggesting that Ca-dependent signaling was required. IL-1-mediated enhancement of cytokine expression was confirmed at the mRNA level by Northern blot and/or RT-PCR analysis. Our study reveals a role for IL-1 in the up-regulation of multiple mast cell-derived cytokines. Moreover, we identify mast cells as a novel source of IL-9. These results are of particular importance in the light of recent reports that strongly support a central role of IL-9 in allergic lung inflammation and in host defense against worm infections.     

Effects of L-leucine methyl ester (Leu-OMe) on mouse peritoneal mast cells: characterization of histamine release versus cytotoxicity.

L-Leucine methyl ester (Leu-OMe), a lysosomotropic compound, has been found to eliminate several lysosome-rich cellular subtypes and all natural killer cell function from peripheral blood mononuclear cells. In this report, the effect of Leu-OMe on mouse peritoneal mast cells is described. The L-Leu-OMe induced the release of histamine from mouse peritoneal mast cells in a dose-dependent manner (0.25 to 3 mM), while its D-stereoisomer had no effect. L-Leu-OMe displayed also a potent histamine release effect on purified mast cells, indicating a direct effect on mast cells. The monitoring of radioactive chromium release versus histamine release showed that both processes may be unrelated for Leu-OMe concentrations inferior to 1.5 mM. At higher doses, L-Leu-OMe, but not its D-stereoisomer, exerted a potent cytotoxic effect on mast cells. The secretory effect of Leu-OMe was temperature- and energy-dependent. Experiments performed in the absence of extracellular calcium and magnesium demonstrated that these divalent cations were not necessary for the Leu-OMe-induced histamine release, and their deprivation even involved a higher histamine release. The secretory characteristics of the Leu-OMe-induced histamine release appeared to be different from those of the IgE-induced ones. These results support the conclusion that exposure of mouse peritoneal mast cells to high doses of L-Leu-OMe results in killing of these cells, that are new targets of this lysosomotropic agent.     

A critical role for mast cells and mast cell-derived IL-6 in TLR2-mediated inhibition of tumor growth

Several TLR agonists are effective in tumor immunotherapy, but their early innate mechanisms of action, particularly those of TLR2 agonists, are unclear. Mast cells are abundant surrounding solid tumors where they are often protumorigenic and enhance tumor angiogenesis. However, antitumor roles for mast cells have also been documented. The impact of mast cells may be dependent on their activation status and mediator release in different tumors. Using an orthotopic melanoma model in wild-type C57BL/6 and mast cell-deficient Kit(W-sh/W-sh) mice and a complementary Matrigel-tumor model in C57BL/6 mice, mast cells were shown to be crucial for TLR2 agonist (Pam(3)CSK(4))-induced tumor inhibition. Activation of TLR2 on mast cells reversed their well-documented protumorigenic role. Tumor growth inhibition after peritumoral administration of Pam(3)CSK(4) was restored in Kit(W-sh/W-sh) mice by local reconstitution with wild-type, but not TLR2-deficient, mast cells. Mast cells secrete multiple mediators after Pam(3)CSK(4) activation, and in vivo mast cell reconstitution studies also revealed that tumor growth inhibition required mast cell-derived IL-6, but not TNF. Mast cell-mediated anticancer properties were multifaceted. Direct antitumor effects in vitro and decreased angiogenesis and recruitment of NK and T cells in vivo were observed. TLR2-activated mast cells also inhibited the growth of lung cancer cells in vivo. Unlike other immune cells, mast cells are relatively radioresistant making them attractive candidates for combined treatment modalities. This study has important implications for the design of immunotherapeutic strategies and reveals, to our knowledge, a novel mechanism of action for TLR2 agonists in vivo.     

泛素、泛素链和蛋白质泛素化研究进展

蛋白质泛素化是以泛素单体和泛素链作为信号分子,共价修饰细胞内其他蛋白质的一种翻译后修饰形式。不同蛋白质底物、同一底物的不同氨基酸修饰位点以及同一位点上泛素链连接方式的不同均可导致细胞效应的差异。蛋白质泛素化在真核细胞内广泛存在,除了介导蛋白质的26S蛋白酶体降解途径之外,还广泛参与了基因转录、蛋白质翻译、信号传导、细胞周期控制以及生长发育等几乎所有的生命活动过程。泛素链的形成及其修饰过程的任何失调均可导致生物体内环境的紊乱,从而产生严重的疾病。文中结合实验室研究,综述了泛素的发现历史、基因特点、晶体结构,特别是泛素链的组装过程、结构、功能以及与人类相关疾病关系的新进展,可为这些疾病的治疗靶点和药物靶标的研究提供思路。     

Redundant and opposing functions of two tyrosine kinases, Btk and Lyn, in mast cell activation

Protein-tyrosine kinases play crucial roles in mast cell activation through the high-affinity IgE receptor (FcepsilonRI). In this study, we have made the following observations on growth properties and FcepsilonRI-mediated signal transduction of primary cultured mast cells from Btk-, Lyn-, and Btk/Lyn-deficient mice. First, Lyn deficiency partially reversed the survival effect of Btk deficiency. Second, FcepsilonRI-induced degranulation and leukotriene release were almost abrogated in Btk/Lyn doubly deficient mast cells while singly deficient cells exhibited normal responses. Tyrosine phosphorylation of cellular proteins including phospholipases C-gamma1 and C-gamma2 was reduced in Btk/Lyn-deficient mast cells. Accordingly, FcepsilonRI-induced elevation of intracellular Ca2+ concentrations and activation of protein kinase Cs were blunted in the doubly deficient cells. Third, in contrast, Btk and Lyn demonstrated opposing roles in cytokine secretion and mitogen-activated protein kinase activation. Lyn-deficient cells exhibited enhanced secretion of TNF-alpha and IL-2 apparently through the prolonged activation of extracellular signal-related kinases and c-Jun N-terminal kinase. Potentially accounting for this phenomenon and robust degranulation in Lyn-deficient cells, the activities of protein kinase Calpha and protein kinase CbetaII, low at basal levels, were enhanced in these cells. Fourth, cytokine secretion was severely reduced and c-Jun N-terminal kinase activation was completely abrogated in Btk/Lyn-deficient mast cells. The data together demonstrate that Btk and Lyn are involved in mast cell signaling pathways in distinctly different ways, emphasizing that multiple signal outcomes must be evaluated to fully understand the functional interactions of individual signaling components.     

B cell stimulatory factor-1/interleukin-4 mRNA is expressed by normal and transformed mast cells

BSF-1/interleukin-4, a product of activated T cells, has multiple biological activities that affect cells of most hematopoietic lineages. Among these is the ability of BSF-1 to costimulate the growth of mast cells and regulate the production of IgE. We demonstrate here that BSF-1 mRNA is expressed by a majority of transformed mast cell lines and by 5 IL-3-dependent non-transformed mast cell lines. BSF-1 activity, including the ability to enhance the growth of IL-3-dependent mast cells, was detected in the supernatants of transformed mast cells. The role of BSF-1 as a mast cell growth factor, its constitutive production by transformed mast cells, and the lack of IL-3 production by most of these cells raise the possibility that BSF-1 may act as an autocrine growth factor for some transformed mast cells. Furthermore, production of BSF-1 mRNA by nontransformed cells indicates that mast cells may be an important physiologic source of this factor.