IgE receptors from rat intestinal mucosal and peritoneal mast cells show mast cell subtype-specific differences

High- (alpha chain) and low-affinity IgE receptors from purified populations of rat intestinal mucosal (IMMC) and peritoneal mast cells (PMC) were characterized by SDS-PAGE. Receptor expression and molecular weight were compared. IMMC yielded 59-kilodalton (kDa) alpha chains of the high-affinity receptors and two forms (58, 50 kDa) of low-affinity receptors, whereas PMC possessed only 51-kDa alpha chains and 56-kDa low-affinity receptors. These differences extend the evidence for functional diversity between mast cell subtypes.     

Isolation and characterization of IgE receptors from rat intestinal mucosal mast cells

High-(Fc epsilon RI) and low-(Fc epsilon RII) affinity IgE receptors were isolated from surface radioiodinated, Nonidet-P40-solubilized rat intestinal mucosal mast cells (IMMC) and compared with those on rat peritoneal mast cells (PMC) and rat basophilic leukemia (RBL) cells. Fc epsilon RII were isolated by affinity chromatography using IgE-Sepharose or by anti-Fc epsilon RII antisera and protein A-Sepharose. The surface-exposed, IgE-binding alpha subunits of Fc epsilon RI [Fc epsilon RI alpha] were isolated by affinity chromatography using IgE and anti-IgE-Sepharose. Fc epsilon RI alpha on IMMC had an apparent molecular mass of 59 kDa, somewhat larger than that of PMC (51 kDa), RBL-2H3 cells (51 kDa) or RBL-CA10.7 cells (46 kDa). Brief (45 s) incubation of IMMC or PMC in glycine-HCl, pH 3, prior to iodination removed much of the surface-bound IgE. This permitted more thorough labeling of the receptors, but had no affect on the estimate of receptor size. Surprisingly and in contrast to acid-treated PMC, upon anti-IgE-Sepharose isolation acid-treated IMMC yielded an intensely radioactive Fc epsilon RI alpha band in the absence of added IgE. Such a finding suggests that IMMC, more so than PMC, may have an intracellular store of IgE, as has been suggested by many others. IMMC also differed from PMC in the number of forms of Fc epsilon RII isolated; 50-kDa and 58-kDa forms of Fc epsilon RII were obtained from IMMC, whereas PMC yielded most often a single 56-kDa Fc epsilon RII band. These results were mimicked by the two RBL cell sublines: RBL-2H3 cells yielded two Fc epsilon RII (46 kDa and 55 kDa), but only one form of Fc epsilon RII (54-kDa) was obtained from RBL-CA10.7 cells. Thus, the two subtypes of rat mast cells, which have previously been shown to differ in mediator profile and responsiveness to secretagogues and antiallergic drugs, are also distinguished by differences in IgER profile.     

Investigations of rat mast cell heterogeneity

In the rat, two distinct mast cell subsets have been identified. One, represented by the easily accessible and purified peritoneal mast cell, differs from the other, the intestinal mucosal mast cell, in staining properties, fixation sensitivity, mediator content, and responsiveness to various secretagogues and antiallergic drugs. To investigate the molecular basis and control mechanisms of mast cell heterogeneity, we have initiated sodium dodecylsulfate polyacrylamide gel electrophoretic analysis of the constituents of rat peritoneal and intestinal mucosal mast cells; developed subcellular fractionation protocols to facilitate these comparisons; produced a mast cell specific rabbit antiserum, and established methods to isolate mast cell RNA. These approaches require refinement, but will prove to be useful in further investigations of mast cell biology.     

Interferons differentially regulate histamine and TNF-alpha in rat intestinal mucosal mast cells.

Mast cell (MC) heterogeneity has been well characterized in the rat where it has been shown that connective tissue MC, often represented by peritoneal MC (PMC), and the intestinal mucosal MC (IMMC) exhibit many differences in mediator content and responsiveness to secretagogues and anti-allergic drugs. Pretreatment (20 hr) of PMC with interferon (IFN)-alpha/beta or IFN-gamma, significantly reduced antigen-stimulated histamine release. By contrast, for IMMC, the same IFN treatment did not modify antigen-stimulated histamine secretion. Although IFN treatment differentially modulates histamine secretion from PMC and IMMC, pretreatment of both MC types with IFN-alpha/beta or IFN-gamma inhibited their tumour-necrosis factor-alpha (TNF-alpha)-dependent cytotoxicity. In 20 hr of culture, IMMC spontaneously released 98 pg/10(6) MC of TNF-alpha, whereas PMC released about threefold more TNF-alpha, 282 pg/10(6) MC. In addition, direct assessment of stored TNF-alpha established that IMMC store less TNF-alpha (68 pg/10(6) MC) than PMC (404 pg/10(6) MC). In summary, TNF-alpha content of PMC and IMMC was different, but IFN inhibited TNF-alpha-dependent cytotoxicity by both MC types. By contrast, treatment with IFN-alpha/beta or IFN-gamma inhibits antigen-induced histamine secretion by PMC, but does not modify antigen-induced histamine secretion by IMMC. Thus, IFN differentially regulate the secretion of histamine and TNF-alpha in PMC and IMMC.     

Factor-independent tissue cultured mast cells: establishment from rat peritoneal mast cells

We report here that extended culture of purified rat peritoneal mast cells (RpMC), typical of the connective tissue-type (CTMC), gives rise to continuously proliferative cell lines without the requirement of exogenous growth factors such as IL-3 and IL-4 or accessory cells. Two of the cell lines established, RCMC1 and RCMC2, are described here. Both cell lines have been maintained in continuous culture in vitro for over a year. Although these cell lines were derived from CTMC, they exhibit phenotypic characteristics of mucosal-type mast cells, i.e., they contain rat mast cell protease II (RMCP II), low levels of histamine and stain alcian blue+/safranin-. Previous studies have identified both high and low affinity receptors for IgE, designated Fc epsilon RI and Rc epsilon RII, respectively, on RpMC and rat basophilic leukemia (RBL) cells. At the early stages of cell culture, RCMC1 expressed predominantly Fc epsilon RI and a gradual increase in the expression of Fc epsilon RII has been observed with time in culture. By comparison, RCMC2 expressed predominantly Fc epsilon RII throughout its entire period of cell culture.     

Mast cell heterogeneity: evidence that mast cells isolated from various connective tissue locations in the rat display markedly graded phenotypes.

The present study has extended histochemical and functional investigations into rat mast cell heterogeneity using isolated mast cells from four connective tissue locations; the peritoneum, mesentery, lung and skin. On histological examination, mast cells from these locations displayed a range of phenotypes following formalin fixation and staining with Alcian blue/safranin O, suggesting the existence of both chondroitin sulphate and heparin proteoglycans in varying proportions in these cell types. Functional studies using the structurally diverse polycationic secretagogues, compound 48/80, the polyamino acids, polymyxin B, substance P, ACTH1-24, mastoparan, protamine sulphate, histone, d-tubocurarine and ranitidine confirmed the existence of such phenotypic gradation. This investigation highlights the inappropriate usage of the terms CTMC and MMC which represent two phenotypic extremes between which a gradation of phenotypes clearly exists.     

Human intestinal mucosal mast cells: evaluation of fixation and staining techniques

The staining properties of tissue mast cells are influenced by the method of fixation. Differences in fixation and staining techniques may explain the contradictory results in the published reports on the number of human mucosal mast cells (MMC) in the gastrointestinal mucosa in health and disease. We have examined the influence of fixatives on the staining properties of human MMC in operative biopsy specimens of human jejunum. Specimens were divided into pieces, each of which was fixed in one of the following fixatives: Carnoy's, basic lead acetate (BLA), Baker's, Bouin's, isotonic formol-acetic-acid (IFAA), 10% neutral buffered formalin, formol sublimate, and formol saline. Thereafter, tissues were paraffin-embedded and 5 micron sections were cut and stained with either astra-blue/safranin pH 0.3, or toluidine blue pH 0.5. Counts of the number of MMC/mm2 were obtained for each fixation method. The results show a critical influence of the fixative on the number of mast cells identified after staining. For example with astra-blue/safranin the mean MMC/mm2 count was 40 in formol-saline-fixed specimens, and 268 in Carnoy's-fixed specimens. In biopsies fixed with formalin-based fixatives, mast cells were more readily stained with toluidine blue. It is recommended that Carnoy's or BLA be used as the fixative for any light microscopic study of human MMC.     

Mast cell heterogeneity in the gastrointestinal tract: variable expression of mouse mast cell protease-1 (mMCP-1) in intraepithelial mucosal mast cells in nematode-infected and normal BALB/c mice.

Soluble granule chymases in rodent intestinal mucosal mast cells (IMMCs) may play an important role in altering epithelial permeability during immediate hypersensitivity reactions. Using a monoclonal antibody against the chymase mouse mast cell protease-1 (mMCP-1), we have shown that it is constitutively expressed in < or = 20% of esterase-positive (esterase+) IMMCs but not in esterase+ gastric mucosal mast cells (GMMCs) in normal BALB/c mice. Intestinal infection with mouse- or rat-adapted strains of Nippostrongylus brasiliensis resulted in IMMC hyperplasia with 100% of esterase+ IMMCs expressing mMCP-1. In contrast, there was a variable response in terms of numbers of GMMCs and of the proportion expressing mMCP-1. Esterase+ mast cells in the gastric submucosa, muscularis, ear pinna, lung parenchyma, major airway submucosa, and peritoneal cavity did not express mMCP-1. The few airway esterase+ mast cells expressing mMCP-1 were, like the great majority of IMMCs and GMMCs, located intraepithelially. In conclusion, mMCP-1 is predominantly expressed by intraepithelial mucosal mast cells but not in all sites; the immunological stimulus associated with intestinal nematodiasis substantially up-regulates mMCP-1 expression by mast cells in the jejunum but not in the stomach; IMMCs and GMMCs in BALB/c mice are phenotypically and possibly functionally distinct.     

Mast cell heterogeneity: Evidence and implications

Mast cells and basophils play a fundamental role in the pathogenesis of allergic disease, although their physiologic role is largely unknown. A large body of evidence now indicates that the properties of mast cells are dependent on the tissue and species from which they are derived. Such mast cell heterogeneity encompasses differences in morphology, development, cytochemistry, and function. The evidence for such heterogeneity, and some of its clinical implications, is discussed.     

Morphological alterations in rat peritoneal mast cells by stem cell factor.

Stem cell factor (SCF) stimulates mast cell adhesion and, because SCF is produced normally in tissues, it may be a major factor responsible for the adhesion of mast cells to connective tissue matrix. We found that the morphology of rat peritoneal mast cells (RPMC) altered after the addition of recombinant murine SCF (rmSCF) in vitro. The ability of rmSCF to enhance morphological alteration was dose dependent and completely abolished by anti-c-kit ACK2 monoclonal antibody. Exposure of RPMC to transforming growth factor-beta 1, wortmannin, genistein, herbimycin A, staurosporine, indomethacin and cytochalasin D before the addition of rmSCF antagonized rmSCF-induced morphological alteration. However, nordihydroguiaretic acid had no effect. Many RPMC appeared to respond also to nerve growth factor (NGF) but the total number of cells with altered morphology was much greater when the culture was stimulated by rmSCF than by NGF. We suggest that morphological alterations of mast cells by rmSCF is an important step for the participation in adhesion to tissue under resident physiological conditions.     

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.     

一种大鼠腹腔肥大细胞分离方法

目的介绍一种密度梯度离心分离大鼠腹腔肥大细胞(RPMC)的技术。方法比较改良方法和原方法2种分离的细胞量、活性及纯度。台盼蓝染色检测细胞存活率;甲苯胺蓝染色检测肥大细胞纯度。将弃去密度梯度离心上液2 m(lA方法)与2.5 m(lB方法)比较,以分析弃去的细胞液的量与肥大细胞纯度的关系。用C48/80刺激分离的RPMC,检测细胞释放的β-氨基己糖苷酶和组胺以确定分离的RPMC脱颗粒活性;透视电镜扫描观察细胞。结果改良方法分离的细胞显著多于原方法分离的细胞数;两种方法分离的细胞存活率都在99%以上;肥大细胞纯度都在90%以上。C48/80刺激RPMC,分离的细胞释放大量的β-氨基己糖苷酶和组胺,高于空白对照组。电镜观察细胞具有明显的肥大细胞形态。结论改良的Percoll密度梯度离心分离大鼠腹腔肥大细胞的方法可行。     

Mechanism of histamine release from rat peritoneal mast cells

Summary The present communication endeavours to elucidate the mechanism of histamine release from rat peritoneal mast cells induced by selective histamine liberators.Of the different enzymatic processes involved in secretion the following are considered: ecto-ATPase activity in the mast cell, pro-esterase-esterase conversion during histamine secretion, cyclic AMP and microtubule association/dissociation, phospholipase A₂ and the effect of phospholipid metabolites on secretion, N-methyl transferase and the methylation of phospholipids and the phosphorylation and desphosphorylation of proteins.     

Effects of oestrogenic agents on rat peritoneal mast cells

Objectives and design:  The objective of this study was to explore whether increased levels of inflammatory cytokines are associated with the risk of clinically silent coronary artery disease. Subjects:  Three-hundred-fifty-six black adults aged 25–54 residing in inner city of Baltimore, Maryland, United States were included in this study. Methods:  Sociodemographics were assessed as were lipid profiles, IL-6, tumor necrosis factor-alpha (TNF-alpha), soluble intercellular adhesion molecule-1 (sICAM-1), and high-sensitivity C-reactive protein (hs-CRP) levels. Computed tomography (CT) coronary angiography was performed. Results:  Coronary calcification was identified in 22.5 % participants and 14 % had significant (≥50 %) coronary stenosis. Multiple logistic regression analyses suggested that IL-6 levels were independently associated with the presence of coronary calcification and significant coronary stenosis, while TNF-alpha, sICAM-1 and hs-CRP levels were not. Conclusions:  This study underscores a critical role for IL-6 in atherosclerosis and suggests that IL-6 may be a marker for significant coronary stenosis in cardiovascularly asymptomatic individuals. This research was funded by National Institute on Drug Abuse grants RO1-DA12777 (Lai S), and RO1-DA15020 (Lai S). Received 22 November 2007; returned for revision 28 May 2008; received from final revision 6 August 2008; accepted by M. Parnham 7 August 2008     

血管活性肠多肽及降钙素基因相关肽对大鼠腹腔肥大细胞组胺释放的作用

目的:研究血管活性肠多肽(Vasoactive intestinal peptide,VIP)及降钙素基因相关肽(Calcitonin gene relatedpeptide,CGRP)对大鼠腹腔肥大细胞脱颗粒的诱导作用;了解神经多肽与肥大细胞的相互关系。方法:分离、纯化SD大鼠腹腔肥大细胞;应用不同浓度的VIP和CGRP作用于大鼠腹腔肥大细胞后,同位素放射液态闪烁法检测肥大细胞的组胺释放、45Ca摄入的变化;同时观察大鼠腹腔肥大细胞经5×10-6mol/L VIP受体抑制剂L-8-K处理后,对VIP诱导脱颗粒作用的影响。结果:5×10-6mol/L的VIP作用后大鼠腹腔肥大细胞组胺释放及45Ca摄入明显增加,并且这种变化与VIP呈剂量效应关系;CGRP对大鼠腹腔肥大细胞组胺释放无诱导作用;L-8-K作用后,肥大细胞对VIP的诱导活化作用无改变。结论:VIP可引起肥大细胞钙内流增加,进一步诱导肥大细胞脱颗粒、释放组胺等生物活性物质,产生生物学效应;这种作用是受体非依赖性的,且与VIP的分子构型有关。     

Desensitization of rat peritoneal mast cells to substance P

Rat peritoneal mast cells were pretreated for 10 min at 37°C with either substance P (SP, 3 or 6 M) or compound 48/80 (37.5, 50 or 75 ng/ml). The effect of this pretreatment on the subsequent responsiveness of the cells to SP was studied. Both SP and compound 48/80 pretreatment of rat peritoneal mast cells inhibited the subsequent response of the cells to SP. The degree of inhibition produced by either SP or compound 48/80 was dependent on the concentration used to pretreat the cells. Inhibition of the response of the cells to SP was observed whether or not the pretreating agent was removed or remained in contact with the cells during the subsequent stimulation with SP. It is concluded that compound 48/80 and SP desensitize the cells to subsequent stimulation by SP and possible mechanisms for this are discussed.