Spontaneous histamine secretion during isolation of rat cardiac mast cells

Objektive: To test the hypothesis that spontaneous release of histamine occurring during an isolation protocol may modify responses of rat cardiac mast cells (connective tissue-type mast cells) to secretagogues.Methods: We assessed two protocols for enzymatic dispersion utilizing collagenase, hyaluronidase, and deoxyribonuclease; with protease (Protocol 1, n = 8) or without protease (Protocol 2, n = 3). Spontaneous release of histamine was quantified following mechanical and enzymatic dispersion of the whole heart.Results: Total histamine loss (Mean ± SEM) was 963±92 and 833±60 ng/g of tissue weight following Protocols 1 and 2. Percentages of histamine release from cell isolates following Protocol 1 were 40±5%, 41±6%, and 51±7% at 0, 30, and 300 ug/mL of compound 48/80.Conclusions: Enzymatic dispersion of cardiac mast cells affects their response to secretagogues.Received 8 January 2004; returned for revision 5 February 2004; accepted by A. Falus 22 March 2004     

A central role for the mast cell in early phase vasculitis in the Brown Norway rat model of vasculitis: a histological study

Administration of mercuric chloride (HgCl(2)) to Brown Norway rats causes Th2-dominated autoimmunity with raised immunoglobulin E concentrations and gut vasculitis, both of which are T-cell dependent, peak at 14 days after starting HgCl(2) and then spontaneously resolve. If animals are re-challenged with HgCl(2) 6 weeks after initial exposure, they are resistant to autoimmunity, developing only attenuated disease. Recently, a separate phase of early caecal vasculitis was described beginning 24 h after initiating HgCl(2) and prior to caecal entry of T cells. Previous work suggested this early vasculitis was alpha beta T-cell independent and implied a role for mast cells. We further tested this hypothesis by performing a histological study during the first 93 h following HgCl(2) challenge defining the precise relationship between gut mast cell degranulation and appearing caecal vasculitis. We also studied whether early caecal vasculitis enters a resistant phase upon re-challenge with HgCl(2). We show a direct correlation between mast cell degranulation and early caecal vasculitis following initial HgCl(2) challenge. We demonstrate resistance to re-challenge in this phase of injury, with results at re-challenge also showing a correlation between mast cell degranulation and early caecal injury.     

Expression and function of fibronectin binding integrins on rat mast cells

Adhesion molecules of the integrin family are implicated notonly in leukocyte migration but also in leukocyte activation.Here we characterize the expression and function of fibronectinreceptor integrins on rat mast cells. A rat basophilic leukemiacell line (RBL-2H3) and phorbol esterstimulated rat peritonealmast cells adhered to fibronectin (FN), vitronectin and fibrinogen.These mast cells expressed fibronectin receptor integrins, Includingvery late antigen (VLA)-4, VLA-5 and vitronectin receptor (VNR),as estimated by immunofluorescent staining and inhibition ofFN adherence by newly established mAbs reactive with the rat4 (MR4-1), 5 (HM5-1) or ß3 (HMß3-1) chainsof the integrin molecules. The ß-hexosaminidase release,a marker for mast cell degranulation, triggered by high affinityIgE receptor (FcRI)-medlated stimulation, was enhanced by adhesionof RBL-2H3 cells to either immobilized FN, MR4-1, HM5-1 or HMß3-1.This FN enhancement of ß-hexosaminidase release wasinhibited by soluble MR4-1, HM5-1 and HMß3-1 as wellas by GRGDSP and DELPQLVTLPHPNHLGPEILDVPST peptides which abrogateVLA-5/VNR and VLA-4 binding to FN respectively. In vivo, passivecutaneous anaphylaxis induced by IgE anti-DNP and DNP-BSA wasinhibited by concurrent s.c. injection of MR4-1, HM5-1 and HMß3-1.These results demonstrate that FN receptor integrins expressedon rat mast cells play an important role in regulating mastcell activation both in vitro and in vivo.     

Myelin basic protein stimulates insulin and glucagon secretion from rat pancreatic islets in vitro and in vivo

The effect of myelin basic protein on insulin and glucagon secretion from rat pancreatic islets was studied in vivo and in vitro. The myelin basic proteins isolated from bovine, human and rat brains all stimulated insulin secretion in a similar fashion. In a static incubation of isolated pancreatic islets, myelin basic protein at doses of 15.6–250 μg in a 0.5-ml reaction volume (1.7 times 10^-8 to 2.7 times 10^-5 M) significantly stimulated hormone release. Maximal stimulation, obtained at the 250-μg dose, was 6.5-fold greater than control for insulin secretion and 6.7-fold greater than control for glucagon secretion. In the case of glucagon no saturation was observed, but saturation was obvious for insulin release at doses of myelin basic protein of 62.5–250μg, larger doses causing permeabilization of the islet membranes as indicated by leakage of acid phosphatase. At a 100-μg dose the time course of insulin secretion induced by myelin basic protein indicated a fast initial release, and after the first 2 h only a little more insulin was released. At the lower doses of myelin basic protein (11 and 33μg) the secretion rate was nearly constant after the first hour. Significant stimulation of glucagon release by myelin basic protein was seen after 60 min, the rate of release being roughly constant at 33-and 100-μg doses thereafter. At the 11-μg dose significant stimulation of hormone release was observed only after a 4-h incubation. Lowering the temperature from 37 to 27 and 20°C reduced both basal and stimulated hormone secretion, the extent of stimulation over the basal level remaining the same at all temperatures only for insulin secretion at a dose of myelin basic protein of 100 μg. A dose of 10 mg myelin basic protein injected intravenously into anaesthetized rats resulted 15 min after injection in a circulating concentration of myelin basic protein of 34.7 μg ml^-1 (1.7 times 10^-6 M) as measured by our radioimmunoassay. It stimulated insulin secretion (P < 0.01), having no significant effect on plasma glucagon levels. Since myelin basic proteins have been shown to display fusogenic properties in cell-free membrane systems, we propose that myelin basic protein exerts its hormone-releasing effect by aggregating and fusing the hormone-containing vesicles to the cell plasma membranes.     

Transmural changes in mast cell density in rat heart after infarct induction in vivo

The cardiac distribution of mast cells was investigated after the induction of acute myocardial infarction in the rat. The left anterior descending coronary artery (LAD) was occluded by ligation in the infarct group, whereas in sham rats only a superficial ligature was placed beside the LAD. Rats of both groups were killed at 4, 7, 14, 21, 35, and 85 days following surgery. Hearts were excised and formalin-fixed. Mast cell densities were monitored in subepicardial and subendocardial layers of the left ventricle (LV) in 6 μm thick toluidine blue-stained cross-sections. In control (non-operated) animals, mast cell densities were comparable in the LV subepicardial and subendocardial layers (1·5–2·0 cells per mm²). Following infarction, the mast cell density at the subepicardial site of the infarction gradually increased, reaching a maximum of 25 cells per mm² on day 21, while a non-significant increase was observed at the subendocardial site. In the non-infarcted regions, the mast cell density increased transiently to reach a maximum of 7 cells per mm² on day 35 in the subepicardial layer. Again, changes in mast cell density in the subendocardial layer were non-significant. In the sham group, a gradual increase to 9 cells per mm² on day 21 and a subsequent decrease to 5 cells per mm² on day 85 were observed in the subepicardial layers. These findings indicate a massive accumulation of mast cells in the subepicardial layers of the infarcted region and a small but significant effect of the surgical procedure on cardiac mast cell deposition, especially in the outer layers of the left ventricle.     

Developmental changes of mast cell populations in the cerebral meninges of the rat

It is known that both the dura and the pia mater attract and support the differentiation of mast cells. The present study shows that unevenly distributed mast cells in the cerebral meninges of the rat can be found in perivascular sites and vessel ramification points, but can also be unrelated to the meningeal vasculature. It also documents changes in the number, localization and staining preferences of the mast cells in the two meninges of the developing and mature rat brain. Quantitative examination of all types of histochemically differentiated meningeal mast cells reveals no major (although some exist) differences between right and left side subpopulations, but strongly suggests a different origin and fate of the dural and the pial mast cells. The number of dural mast cells, already high from postnatal day 0, although declining from postnatal day 21 onwards, remains conspicuous up to postnatal day 180. In contrast, pial mast cells are comparatively very few in the first day of the postnatal life, and despite a transient significant increase in the following two weeks, they reach almost zero levels from postnatal day 21.     

IgA肾病患者肾间质肥大细胞浸润的临床意义

目的 研究肥大细胞（MC）在IgA肾病患者肾间质中的分布及间质纤维化的关系。方法 采用甲苯蓝特殊染色法及MC特异酶（即类酶蛋白酶,tryptase）免疫组化染色法,对12例IgA肾病患者肾活检标本中的MC进行了观察。结果 （1）肾脏皮、髓质均可见到MC,多见于纤维化区域、血管周围、萎缩或扩张小管周围及肾小球周围。（2）系膜细胞的增殖程度与MC数无关,而随着IgA肾病患者MC的数增加,间质纤维化加重,差异显著（P＜0．05）。结论 IgA肾病患者的肾间质中存在MC,并与肾间质的纤维化有关。     

Histamine-liberating action of antihistamines on isolated rat mast cells

Of the new antihistamines (quinuclidine derivatives) tested, quinuclidyl-3-(o-tolyl) carbinol possessed histamine-liberating activity (HLA) on isolated rat mast cells. In concentrations tested (up to 0.4 mM) all phenothiazines (promethazine, phenethazine, chlorpromazine, methylene blue) possessed HLA. No connection was found between HLA and the antihistamine action of the compounds tested. Histamine liberation induced by antihistamines had a steep dose-response curve, took place at a low temperature, and was not inhibited under conditions inhibiting the energydependent stage of histamine liberation induced by compound 48/80. It is concluded that the antihistamines tested and found to possess HLA are nonselective liberators of histamine.Allergologic Research Laboratory, Academy of Medical Sciences of the USSR. Laboratory of Pharmacology, S. Ordzhonikidze Pharmaceutical Chemical Research Institute, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 3, pp. 329–332, March, 1978.     

大鼠肝内移植肿瘤与肥大细胞关系的形态学研究

目的：研究大鼠肝内移植肿瘤与其周边肥大细胞的关系。方法：建立40只雄性Wistar大鼠肝内移植肿瘤模型，运用HE染色，肥大细胞（mast cell，MC）Alcian blue特殊染色，苦味酸-天狼猩红染色和电镜等技术，观察移植肿瘤肝组织的形态学改变，肿瘤周边浸润MC的数量以及MC与肿瘤组织的关系，8只正常大鼠为对照组。结果：肿瘤组织周边部分肝细胞形态学异常；肝组织内胶原纤维增生，主要为Ⅰ型和Ⅲ型胶原，并包绕肿瘤组织，肿瘤周边MC浸润，并沿胶原纤维分布，不同大鼠其肿瘤周边MC数量不一，有的差异明显。超微结构观察显示MC通过多个接触点与肿瘤细胞紧密相连，并释放胞质颗粒内容物，导致肿瘤细胞崩解。结论：MC与肿瘤组织有着非常密切的关系，MC可能通过多种途径直接和间接地发挥抗肿瘤作用。     

Juxtacellular histamine concentration governs histamine release from rat peritoneal mast cells

Isolated rat peritoneal mast cells release histamine when superfused with isoosmotic salt or sucrose solutions. The release was ascribed by us to an intracellular ion exchange between potassium and histamine at granule sites, resulting from a flux of cytoplasmic potassium across the granules secondary to the disturbance of the ‘state of equilibrium’ at the cell surface caused by the superfusion (Uvnäs et al. 1989). In the present article is shown that the histamine releasing effect is counteracted by the addition of histamine to the superfusion fluid. The inhibition is concentration-dependent and accompanied by concomitant changes in the potassium efflux. A 50% inhibition of the histamine release requires an external histamine concentration of 40 μm and extrapolation of the equilibrium curve hints at a total inhibition at concentrations around 170 μm. The observations are taken to indicate that reduction of the juxtacellular histamine concentration caused by the superfusion disturbs the histamine equilibrium at the mast cell surface resulting in the activation of the histamine secretory mechanism. In other words, the secretory activity of the mast cell is checked by the juxtacellular concentration of histamine. When the juxtacellular histamine is removed e.g. on isolation procedures, other experimental situations such as superfusion, or by consumption in vivo the mast cell delivers histamine to restore the juxtacellular equilibrium.     

Recombinant human alpha-2a interferon promotes an atypical process of mast cell secretion with ultrastructural features suggestive for piecemeal degranulation

Purified mast cells (MC), isolated from the rat peritoneum, were stimulated in vitro with recombinant human IFN-alpha 2a (rhIFN-alpha 2a) and studied ultrastructurally. Quantitative determination of histamine release was also performed. The following ultrastructural features were observed: (1) dilation of single granules, leading to the formation of cytoplasmic cavities filled with dissolved or eroded matrices; (2) induction of partially empty, nonfused granule containers close to unaltered resting granules, a process very suggestive of piecemeal degranulation; (3) focal exocytosis, characterized by opening of single granules to the cell exterior and/or fusion of a few granules into small secretory channels. Histamine release was slightly increased in rhIFN-alpha 2a-treated MC, although not to significant levels. These results indicate that rhIFN-alpha 2a induces a characteristic pattern of degranulation in rat peritoneal MC and that a small proportion of rhIFN-alpha 2a-stimulated MC shows ultrastructural features suggestive for piecemeal degranulation.     

腹膜炎大鼠肥大细胞及间皮的形态变化

目的：探讨腹膜炎大鼠间皮细胞的形态变化和肥大细胞的作用。方法：将粪汁注入大鼠腹腔,制成腹膜炎模型。注射后第1、2、3、4h取肠系膜铺片,硫堇染色,光镜观察肥大细胞的数量和形态。间皮细胞的形态做了扫描电镜观察。结果：在腹膜炎的第1～4h,5mm^2肠系膜内可辨认的肥大细胞总数由平均37．7个减至平均13．7个;正在脱颗粒的肥大细胞,由平均1个增至20．8个;核周有少数颗粒的裸核肥大细胞由无增至平均6．1个。间皮细胞的损伤随腹膜炎的发展而加重。结论：肥大细胞释放活性物质,以速发超敏反应参与腹膜炎的病理过程,早期减少腹膜间皮表面积,减少毒素吸收;晚期加速腹膜间皮破坏,增加毒素吸收。     

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.     

Release of somatostatin,neurotensin and vasoactive intestinal peptide upon inhibition of gastric acid secretion by duodenal acid and hyperosmolal solutions in the conscious rat

The inhibitory effect of duodenal exposure to acid and hyperosmolal solutions on pentagastrin-stimulated gastric acid secretion was studied in conscious rats equipped with chronic gastric fistula and duodenal Thiry-Vella loop. The loop was challenged with saline, HCl or hyperosmolal polyethylene glycol. Gastric acid secretion was measured in samples from the gastric fistula. Gut peptide concentrations were measured in duodenal perfusates collected each 30 min, and in plasma samples collected both during stimulated acid secretion alone, and at the end of experiments in combination with luminal challenges of the loops. During pentagastrin-stimulated gastric acid secretion, luminal perfusion of the duodenal loop with acid caused inhibition of acid secretion (P < 0.001) and a prominent release of somatostatin both to the lumen (P < 0.001) and to the circulation (P < 0.05). Also, neurotensin (P < 0.01) and vasoactive intestinal peptide (P < 0.01) were released to the lumen, but not to the circulation. Upon perfusion of the duodenal loop with hyperosmolal polyethylene glycol, acid secretion was inhibited (P < 0.05) and somatostatin alone was released to the luminal side (P < 0.01). In conclusion, duodenal exposure to acid inhibits pentagastrin-stimulated gastric acid secretion and releases SOM to the circulation that may directly inhibit acid secretion. Concomitantly, somatostatin (SOM), neurotensin and vasoactive intestinal peptide are released to the lumen. Duodenal exposure to hyperosmolal polyethylene glycol inhibits acid secretion with a luminal release of SOM only. Thus, luminal acid and hyperosmolal solutions inhibit gastric acid secretion by separate mechanisms. After acid or hyperosmolal challenge, the release of SOM to the circulation indicates gastric acid inhibition in an endocrine manner, while a luminal release of gut peptides indicates a local peptide overflow that might be of importance via paracrine regulatory mechanisms in the intact animal.     

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.