FcγR-dependent apoptosis regulates tissue persistence of mucosal and connective tissue mast cells

Rodent mast cells can be divided into two major subtypes: the mucosal mast cell (MMC) and the connective tissue mast cell (CTMC). A decade-old observation revealed a longer lifespan for CTMC compared with MMC. The precise mechanisms underlying such differential tissue persistence of mast cell subsets have not been described. In this study, we have discovered that mast cells expressing only one receptor, either FcγRIIB or FcγRIIIA, underwent caspase-independent apoptosis in response to IgG immune complex treatment. Lower frequencies of CTMC in mice that lacked either FcγRIIB or FcγRIIIA compared with WT mice were recorded, especially in aged mice. We proposed that this paradigm of FcγR-mediated mast cell apoptosis could account for the more robust persistence of CTMC, which express both FcγRIIB and FcγRIIIA, than MMC, which express only FcγRIIB. Importantly, we reproduced these results using a mast cell engraftment model, which ruled out possible confounding effects of mast cell recruitment or FcγR expression by other cells on mast cell number regulation. In conclusion, our work has uncovered an FcγR-dependent mast cell number regulation paradigm that might provide a mechanistic explanation for the long-observed differential mast cell subset persistence in tissues.     

The role of galanin in the differentiation of mucosal mast cells in mice

Mast cells are generally classified into two phenotypically distinct populations: mucosal-type mast cells (MMCs) and connective tissue-type mast cells (CTMCs). However, the molecular basis determining the different characteristics of the mast cell subclasses still remains unclear. Unfortunately, the number of mast cells that can be obtained from tissues is limited, which makes it difficult to study the function of each mast cell subclass. Here, we report the generation and characterization of MMCs and CTMCs derived from mouse BM mast cells (BMMCs). We found that the expression of galanin receptor 3 was elevated in MMCs when compared to the expression in CTMCs. Moreover, intraperitoneal injection of a galanin antagonist reduced MMCs and inhibited the inflammation of dextran sodium sulfate-induced colitis in mice. Therefore, these results suggest that galanin promotes MMC differentiation in vivo, and provide important insights into the molecular mechanisms underlying the differentiation of mast cell subclasses.     

Multiple Loss of Effector Cell Functions in FcRγ-Deficient Mice

Immunoglobulin Fc receptor (FcR) γ subunit is a component of low affinity receptor for IgG, FCγRIII, as well as high affinity receptor for IgE, FcεRI. This subunit is required for efficient surface expression of these FcRs on various cells in immune system. The FcRγ-deficient mice, generated by gene targeting in embryonic stem cells, exhibit multiple defects in FcR-mediated effector cell responses, including absence of phagocytic activity against opsonized red blood cells by activated macrophages, loss of antibody-dependent cell-mediated cytotoxicity manifested by IL-2-induced splenic NK cells, and unresponsiveness of mast cells to crosslinking of IgE on these cells. These results demonstrate an indispensable role of FcRγ for functional expression of FcRs, and clearly indicate the importance of FCγRIII as well as FcεRI for these effector functions. Since FcRγ-deficient mice is unable to mount the type II and type III hypersensitivity reactions, it is suggested that FcRs play pivotal roles in initiating these reaction cascades. The mutant mice should prove to be useful in evaluating FcRs in various humoral and cellular immune responses, and in developing new strategies for treatment of immunodeficiency as well as autoimmune disorders.     

Role of c-kit receptor tyrosine kinase in the development, survival and neoplastic transformation of mast cells

The c-kit gene is allelic with the dominant spotting ( W ) locus on mouse chromosome 5 and encodes a receptor tyrosine kinase. The llgand for c-klt receptor is stem cell factor (SCF), which is the principal growth factor for mast cells. The loss-of-function mutations of c-kit receptor affect the development of mast cells, thereby resulting in a depletion of mast cells. The abundant expression of c-ktt receptor is indispensable for the survival of mast cells. In addition, the galn-of-function mutations of c-kit receptor were found in several tumor mast cell lines. When these galn-of-function mutations were introduced to cells of murine interleukin (IL)-3-dependent cell lines, the expression of c-kit receptor with constitutive tyrosine kinase activity not only abrogated the IL-3 requirement of the cells, but also caused them to become tumorlgenic in nude athymic mice. The gain-of-function mutations of c-kit receptor appear to result in the malignant transformation of mast cells. Taken together, the signals from the c-ktt receptor are essential for the development, survival, and malignant transformation of mast cells.     

Mast cell activation syndrome—anesthetic challenges in two different clinical scenarios

Mast cell activation syndrome (MCAS) includes a group of disorders that result in the inappropriate release of inflammatory mediators from mast cells. These mediators can affect multiple organ systems and lead to significant morbidity, and possible fatality. Although reactions, typically in response to various nonspecific stimuli, are usually mild, they may put those with MCAS at increased risk of anaphylaxis. In this case report, we present two clinical scenarios of MCAS, and identify possible factors triggering mast cell mediator release. We also define a preoperative preventive pathway, outline anesthetic considerations, and discuss the management of immediate hypersensitivity reactions in patients with MCAS. Meticulous preoperative preparation, avoidance of triggers, and development of a plan to treat possible adverse organ responses are paramount of good outcomes.     

Human bone marrow mast cells in systemic mast cell disease

In a case of systemic mast cell disease with moderate bone marrow involvement, we studied the sensitivity of mast cell cationic dye-binding to formaldehyde fixation, as well as the mast cell ultrastructure, so as to determine whether these cells possess characteristics of mucosal mast cells. This histochemical method has proved to be one of the few which reveal heterogeneity of the mast cells in human intestine. Even though rat bone marrow mast cells have been shown to belong to the mucosal mast cell compartment, we have found no difference in mast cell counts in samples fixed either in formaldehyde or Carnoy's fixative. The ultrastructure did not show major differences with cutaneous mast cells. A few cells presented two nuclei, suggesting mitotic division of mature mast cells in bone marrow.     

肥大细胞在食管癌的特征—组织化学与电镜的观察

对１８例食管癌的肥大细胞进行了组织化学与电镜下的观察，发现在癌组织边缘，癌组织内血管附近结缔组织中及癌旁组织的上皮下与粘膜下层都出现了大量肥大细胞。这些肥大细胞在Ａｌｃｉａｎ蓝，藏红染色，其颗粒主要为Ａｌｃｉａｎ蓝阳性。在显示肝素的硫酸小檗碱荧光反应，在癌组织及旁组织的肥大细胞大多都是阴性。有极少数的癌旁组织的肥大细胞呈黄色荧光反应。电镜下，肥大细胞颗料中具有Ｔ肥大细胞特征的多个涡卷状结构。有些肥     

Inhibition of pathologic inflammation by leukocyte Ig-like receptor B4 and related inhibitory receptors

Summary:  Leukocyte immunoglobulin (Ig)-like receptor B4 (LILRB4)(previously termed gp49B1) is a member of the Ig superfamily expressed constitutively on the surface of mast cells, neutrophils, and macrophages. LILRB4 inhibits IgE-dependent activation of mast cells in vitro through its two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that recruit the src homology domain type-2-containing tyrosine phosphatase 1 into the cell membrane. Accordingly, Lilrb4^−/− mice exhibit greater incidence and severity of IgE- and mast cell-dependent anaphylactic inflammation compared with mice that express LILRB4. In addition, mast cell-dependent inflammation induced by the interaction of stem cell factor (SCF) with its receptor Kit is also more severe in Lilrb4^−/− mice, indicating that the counterregulatory function of LILRB4 extends beyond inflammation induced by Fc receptors, which signal through ITIMs, to responses initiated through a receptor tyrosine kinase. Indeed, pathologic inflammatory responses induced by activation of neutrophils with lipopolysaccharide (LPS) alone or with tissue-specific autoantibodies are greatly exacerbated in Lilrb4^−/− mice. The rapid upregulation of LILRB4 expression on neutrophils in Lilrb4^+/+ mice in response to LPS suggests it is an innate counterregulatory response designed to reduce pathologic inflammation. Nevertheless, LILRB4 also serves a similar purpose for inflammation induced by the humoral adaptive immune response that is manifested through effector cells bearing Fc receptors.     

Protein tyrosine kinase p53/p56(lyn) forms complexes with gamma-tubulin in rat basophilic leukemia cells.

The aggregation of receptors with high affinity for IgE (FcepsilonRI) on the surface of mast cells and basophils initiates a chain of biochemical events culminating in the release of allergy mediators. Although microtubules have been implicated in the activation process, the molecular mechanism of their interactions with signal transduction molecules is poorly understood. Here we show that in rat basophilic leukemia cells large amounts of alphabeta-tubulin dimers ( approximately 70%) and gamma-tubulin ( approximately 85%) are found in a soluble pool which was released from the cells after permeabilization with saponin, or extraction with non-ionic detergents. Soluble tubulins were found in large complexes with other molecules. Complexes of soluble gamma-tubulin released from activated cells contained tyrosine-phosphorylated proteins of relative mol. wt approximately 25, 50, 53, 56, 60, 75, 80, 97, 115 and 200 kDa. Increased tyrosine phosphorylation of proteins associated with the cytoskeleton, i.e. around centrosomes, was detected by immunofluorescence microscopy. In vitro kinase assays revealed increased tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from activated cells. Two of the tyrosine phosphorylated proteins in these complexes were identified as the p53/56(lyn) kinase. Furthermore, gamma-tubulin bound to the N-terminal fragment of recombinant Lyn kinase and its binding was slightly enhanced in activated cells. Pretreatment of the cells with Src family-selective tyrosine kinase inhibitor, PP1, decreased the amount of tyrosine phosphorylated proteins in gamma-tubulin complexes, as well as the amount of gamma-tubulin in Lyn kinase immunocomplexes. The combined data suggest that gamma-tubulin is involved in early stages of mast cell activation.     

Pronociceptive response elicited by TRPA1 receptor activation in mice

Ankyrin-repeat transient receptor potential 1 (TRPA1) is a member of the transient receptor potential (TRP) channel family and it is found in sensory neurons. In the present study, we found that TRPA1 receptor activation with allyl isothiocyanate or cinnamaldehyde caused dose-dependent spontaneous nociception when injected into the mouse hind paw. Very similar results were obtained when stimulating transient receptor potential vanilloid 1 (TRPV1) receptors with capsaicin. Pretreatment with the TRP receptor antagonist Ruthenium Red (1 nmol/paw) inhibited capsaicin-(0.1 nmol/paw) and allyl isothiocyanate-(1 nmol/paw) induced nociceptive responses. However, the nonselective TRPV1 receptor antagonist capsazepine (1 nmol/paw) and the selective TRPV1 receptor antagonist SB 366791 (1 nmol/paw) only attenuated capsaicin-induced nociception. In contrast, the intrathecal treatment with TRPA1 antisense oligodeoxynucleotide (2.5 nmol/site) and the degeneration of the subset of primary afferent fibers sensitive to capsaicin significantly reduced allyl isothiocyanate-induced nociception. Consequently to TRPA1 antisense oligodeoxynucleotide treatment there was a marked decrease of the expression of TRPA1 receptor in both sciatic nervous and spinal cord segments. Moreover, capsaicin and allyl isothiocyanate-induced nociception were not significantly changed by chemical sympathectomy produced by guanethidine. The previous degranulation of mast cells by compound 48/80 and treatment with antagonist H(1) receptor antagonist pyrilamine (400 microg/paw) both significantly inhibited the capsaicin- and allyl isothiocyanate-induced nociception. The selective NK(1) receptor antagonist N(2)-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl) carbony-1-L-prolyl]-N-methyl-N-phenylmethyl-3-2-(2-naphtyl)-L-alaninamide (10 nmol/paw) reduced either capsaicin- or allyl isothiocyanate-induced nociception. Collectively, the present findings demonstrate that the TRPA1 agonist allyl isothiocyanate produces a consistent nociceptive response when injected into the mouse paw, an effect that seems to be mediated via activation of TRPA1 receptor and dependent on the capsaicin-sensitive fibers, release of histamine by mast cells and participation of tachykinins. Thus, the TRPA1 receptor has an apparently relevant role in nociceptive processes and the selective TRPA1 antagonist might possess a potential antinociceptive property.     

Committed mast cell progenitors in mouse blood differ in maturity between Th1 and Th2 strains

Mast cell progenitors (MCp) leave the bone marrow and migrate to peripheral tissues where they mature. Although the existence of committed MCp in adult mouse and human blood has been postulated, they have never been found. We have isolated a rare population of cells in adult mouse blood, committed to the mast cell lineage. These were identified as lineage⁻ c‐kit^hi ST2⁺ integrin β7^hi CD16/32^hi cells. Moreover, a major difference in maturity of these cells based on FcεRI expression was observed between the Th2‐prone BALB/c strain and the Th1‐prone C57BL/6 strain (66% vs 25% FcεRI⁺, respectively). Therefore, the choice of mouse strain is critical when studying disease models such as experimental asthma where mast cells and their progenitors are involved.     

Changing the threshold—Signals and mechanisms of mast cell priming

Mast cells play a key role in allergy and other inflammatory diseases involving engagement of multivalent antigen with IgE bound to high-affinity IgE receptors (FcεRIs). Aggregation of FcεRIs on mast cells initiates a cascade of signaling events that eventually lead to degranulation, secretion of leukotrienes and prostaglandins, and cytokine and chemokine production contributing to the inflammatory response. Exposure to pro-inflammatory cytokines, chemokines, bacterial and viral products, as well as some other biological products and drugs, induces mast cell transition from the basal state into a primed one, which leads to enhanced response to IgE-antigen complexes. Mast cell priming changes the threshold for antigen-mediated activation by various mechanisms, depending on the priming agent used, which alone usually do not induce mast cell degranulation. In this review, we describe the priming processes induced in mast cells by various cytokines (stem cell factor, interleukins-4, -6 and -33), chemokines, other agents acting through G protein-coupled receptors (adenosine, prostaglandin E₂, sphingosine-1-phosphate, and β-2-adrenergic receptor agonists), toll-like receptors, and various drugs affecting the cytoskeleton. We will review the current knowledge about the molecular mechanisms behind priming of mast cells leading to degranulation and cytokine production and discuss the biological effects of mast cell priming induced by several cytokines.     

A novel lipid raft-associated glycoprotein, TEC-21, activates rat basophilic leukemia cells independently of the type 1 Fc epsilon receptor.

Recent data suggest that initiation of signal transduction via type 1 Fc epsilon receptor (Fc epsilon RI) and other immunoreceptors is spatially constrained to lipid rafts. In order to better understand the complexity and function of these structures, we prepared mAb against lipid rafts from the rat basophilic leukemia cell line, RBL-2H3, which is extensively used for analysis of Fc epsilon RI-mediated activation. One of the antibodies was found to recognize a novel glycosylphosphatidylinositol-anchored plasma membrane glycoprotein of 250 amino acids, designated TEC-21, containing a cysteine-rich domain homologous to those found in the urokinase plasminogen activator receptor/Ly-6/snake neurotoxin family. TEC-21 is abundant on the surface of RBL-2H3 cells (>10 (6) molecules/cell), but is absent in numerous rat tissues except for testes. Aggregation of TEC-21 on RBL-2H3 cells induced a rapid increase in tyrosine phosphorylation of several substrates including Syk kinase and LAT adaptor, calcium flux, and release of secretory components. Similar but more profound activation events were observed in cells activated via Fc epsilon RI. However, aggregation of TEC-21 did not induce changes in density of IgE-Fc epsilon RI complexes, tyrosine phosphorylation of Fc epsilon RI beta and gamma subunits, and co-aggregation of Lyn kinase. TEC-21-induced activation events were also observed in Fc epsilon RI(-) mutants of RBL-2H3 cells. Thus, TEC-21 is a novel lipid raft component of RBL-2H3 cells whose aggregation induces activation independently of Fc epsilon RI.     

Diacylglycerol kinases in immune cell function and self-tolerance

Summary: Both diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers involved in signal transduction from many immune cell receptors and can be generated and metabolized through multiple mechanisms. Recent studies indicate that diacylglycerol kinases (DGKs), the enzymes that catalyze phosphorylation of DAG to produce PA, play critical roles in regulating the functions of multiple immune cell lineages. In T cells, two DGK isoforms, α and ζ, inhibit DAG-mediated signaling following T-cell receptor engagement and prevent T-cell hyperactivation. DGK α and ζ synergistically promote T-cell anergy and are critical for T-cell tolerence. In mast cells, DGKζ plays differential roles in their activation by promoting degranulation but attenuating cytokine production following engagement of the high affinity receptor for immunoglobulin E. In dendritic cells and macrophages, DGKζ positively regulates Toll-like receptor-induced proinflammatory cytokine production through its product PA and is critical for host defense against Toxoplama gondii infection. These studies demonstrate pivotal roles of DGKs in regulating immune cell function by acting both as signal terminator and initiator.     

肥大细胞与成纤维细胞功能关系的光镜与电镜研究

通过对正常人体皮肤真皮与胶原纤维大量增生的疾患的真皮组织的光镜与电镜的比较观察,发现在病因不同,而都有胶原纤维大量增生的系统性硬皮病与皮肤纤维瘤的真皮组织中,肥大细胞都比正常时显著增多(P<0.01),而且肥大细胞常与成纤维细胞紧密相靠接,这里的肥大细胞脱颗粒现象也增多。从颗粒的超微结构特征所示,这些肥大细胞以未成熟的T型肥大细胞为主。以上提示:肥大细胞在调节成纤维细胞产生胶原纤维的功能上具有重要意义。     

Expression of the c-kit gene product in normal and neoplastic mast cells but not in neoplastic basophil/mast cell precursors from chronic myelogenous leukaemia

The expression of the c-kit gene product has been examined in normal mast cells, mast cell neoplasms, and basophil/mast cell precursors obtained from patients with chronic myelogenous leukaemia (CML). Formalin-fixed, paraffin-embedded sections or smears fixed with formalin vapour were studied by immunohistochemical methods, using a polyclonal antibody against the c-kit gene product. Normal and neoplastic mast cells showed a positive immunoreaction for c-kit gene product, but neoplastic basophil/mast cell precursors from CML patients lacked c-kit gene product by immunohistochemical and flow cytometric methods, even in cells having mast cell granules, together with or without basophil granules. Mast cell tryptase was, however, expressed in normal and neoplastic mast cells and basophil/mast cell precursors containing mast cell granules. In addition, cells of monocyte/macrophage lineage lacked c-kit gene product. These findings indicate that the c-kit gene product may play an important role in the development and function of mast cell but not of cell of basophil and monocyte/macrophage lineage.     

人腭扁桃体肥大细胞的性质—免疫组化法研究

收集手术切除的人腭扁桃体标本６例，常规石蜡切片，分别以血管活性肠肽、阿尔新蓝－番红花红、长时间甲苯胺蓝和普通甲苯胺蓝染色观察肥大细胞。结果显示：肥大细胞呈血管活性肠肽免疫反应阳性，分布于淋巴小结中央及周围的弥散淋巴组织、被膜及小梁的结缔组织、固有层和小血管周围。阿尔新蓝－番红花红染色呈浅蓝色颗粒。长时间甲苯胺蓝染色显示的肥大细胞数目较多，颗粒为深蓝色。普通甲苯胺蓝染色未以显示肥大细胞。结果提示，人