Heparin secretion by mast cells as an index of the status of the anticoagulant system

The status of the mast cell population was studied and compared after administration of trypsin or alpha-thrombin in similar molar concentrations. Morphometry disclosed a substantial shift of the mast cell population towards light, heparin-free cells within one minute after alpha-thrombin administration. The index of mast cell saturation with heparin dropped below 1. The maximal heparin secretion was observed at the 5th minute of experiment. The morphometric criteria of the mast cell population returned to basal level in 120 minutes. These data along with a significant increase in the level of complex heparin compounds and plasma thrombin time indicate heparin release as a result of the effector action of the anticoagulation system. No changes were observed in the activity of complex heparin compounds and in thrombin time after intravenous injection of trypsin. It is suggested that high heparin secretion by mast cells may serve as criterion of the active status of the anticoagulation system.     

Effect of trophoblast-specific beta 1-glycoprotein on the ratio of cellular forms in a mast cell population

The influence of trophoblast-specific beta 1-glycoprotein (TSG) on the degranulation of mast cells and their saturation with heparin was studied. Introduction of the TSG into the population of mast cells of the rat peritoneal fluid practically does not change their degranulation, but lowers the degree of their saturation with heparin. An antibiotic alone increases the saturation of the cells with heparin. The serum of an allergic animal markedly stimulates the degranulation and lowers the degree of saturation of the mast cells with heparin. In an experimental model (antibiotic--the serum of the allergic mast cells) the mast cells transform into very clear (heparin-free) cells and the degree of saturation is at minimum. The TSG introduction into this system stabilizes the population of mast cells and markedly increases the degree of their saturation with heparin. Although the degranulation is rather intensive, it is less expressed, than in the experimental model. This suggests the presence of TSG receptors on the mast cells (targets of allergic reactions). The possibility to use TSG preparations in the therapy of allergic diseases is discussed.     

Effect of non-fractionated and low-molecular heparin on the functional state of mast cells]

The state of the mast-cell population of rats treated with unfractionated and low-molecular weight heparins under stress conditions has been comparatively studied by the morphometrical assay. The stress was produced by 60 min immobilization followed by intravenous injection of unfractionated (UF) or low-molecular weight (LMW) heparin. The stress-induced heparin release from mast cells resulted in a 3.3-fold decrease of the index of saturation with heparin and in a significant increase of granulolysis and degranulation. The mast cell secretory status reached the preinjection level within 20 min in rats with UF heparin injected (15 unit/200 g). At the same time mast cells of rats with LMW heparin have no such ability. The data obtained indicate that LMW heparin in contrast to UF heparin cannot be accumulated (or accumulated very slowly) by mast cells. This fact as well as low affinity of LMW heparin to endothelium and blood platelets promote its preservation in blood for a long time.     

Effect of exogenous heparin on secretory status of rat mast cells under immobilization stress]

The significant increase of heparin release from mast cells was observed in rats under stress conditions induced by 60 min immobilization. The index of its saturation with heparin became 4 times lower. The highest secretory activity of mast cells was observed during the first 30 min of immobilization. It was shown that at that time the heparin release from mast cells occurred by granulolysis (merocrine type of secretion). In the rats received heparin (15 or 150 u/200 g) during the first 15 min of immobilization the mast cells released heparin with the same intensity as in a 4 control animals. But then in rats with high heparin blood concentration the heparin release from mast cells ceased and mast cells began to accumulate heparin from blood. By the 30th min of immobilization the heparin content in the mast cells has become normal.     

The reaction of the mast cell population to the intratracheal administration of heparin and fucoidan]

Mast cell population of rat after single intratracheal injection of heparin and fukoidan was investigated with cytofluorometry and cytochemical methods. These polysaccharides have great differences in the molecular weight nevertheless they both were found to include in mast cells.     

Mast cell heterogeneity between two different species of Hoplias sp. (Characiformes: Erythrinidae): response to fixatives, anatomical distribution, histochemical contents and ultrastructural features

Mast cells from two erythrinid species: Hoplias malabaricus and Hoplias lacerdae, were studied in several tissues throughout the body using light and electron microscopy. Mast cells were found in all organs studied, but were especially abundant in the gastrointestinal tract, and were always in association with connective tissue. These cells showed different characteristics between the two species studied, like varied morphology, anatomical distribution, density, basophilic/eosinophilic staining and heparin content. In H. malabaricus, the tissues fixed with Helly's solution contained mast cells that were basophilic, metachromatic and had heparin in their cytoplasmic granules, while the tissues fixed with Karnovsky's solution contained eosinophilic and orthochromatic mast cells in which heparin was not detected. In H. lacerdae, the use of both fixatives resulted in mast cells that were eosinophilic, orthochromatic, with no identifiable heparin content. Exclusively in H. malabaricus oesophagus, the mast cells were additionally seen among the epithelial cells. The ultrastructural studies performed in hindgut fixed with Karnovsky's solution revealed that the cytoplasmic granules seen in H. lacerdae mast cells were better preserved than in H. malabaricus mast cells. The latter had electron-lucent granules that were often merged, forming channels. The present study demonstrated that mast cells from two species belonging to the same genus or even mast cells from the same species but under different fixatives can present heterogeneous characteristics, possibly due to their functional properties or to their sensitivity to fixatives.     

Quantitation of mast cell heparin by flow cytofluorometry.

Mast cells can be automatically identified in a mixed cell population by flow cytofluorometry after Berberine sulphate staining. Volume specific counts of the total number of cells and number of mast cells, as well as frequency distributions of fluorescence intensities of mast cells, based on a large number of cells, can be rapidly obtained. Results obtained by microscope fluorometry of cells identified by phase contrast microscopy showviously published results it may be inferred that the fluorescence intensity of individual mast cells is proportional to mast cell heparin content. The automated cell counts correlated very well with manual hemocytometer counts. Both cell counts and the determination of mean mast cell fluorescence showed excellent reproducibility.     

A quantitative cytochemical study of the growth of individual mast cells

Summary For individual mast cells, relationships between their dry mass and their content of heparin and 5-hydroxytryptamine (5-HT) were studied. This was achieved by measuring these parameters successively on identical cells, by means of quantitative cytochemical techniques. The peritoneal mast cells have a very long life span and a slow turnover of granule components. Increase of the dry weight of the cells may therefore be taken as an expression of cellular growth. Mast cell populations from younger and older animals were analysed in an attempt to evaluate the influence of cell-aging and animal-aging on the growth of the mast cells. The analysis was based on allometric (log-log) plots and linear regressions. Within the cell populations there were strong mutual correlations between the cell parameters studied, without any obvious deviations from linearity. However, the slopes of the allometric lines indicated a somewhat different mode of growth for mast cells from younger and older animals. The capacity of the mast cells to accumulate 5-HT after a single injection of its precursor, 5-hydroxytryptophan, was used as a functional test. In relation to the cell weight, the induced increase of 5-HT was greater for lighter than for heavier mast cells. This difference between light and heavy mast cells was greater for cells from younger than from older animals. These differences in growth and functional properties between mast cells from younger and older animals were interpreted as an effect of aging of the animals rather than of aging of the cells.Supported by grants from the Swedish Medical Research Council, Project no 2235I wish to express my sincere thanks to Professor Lennart Enerbäck for valuable and constructive criticism of this paper. I also wish to thank Lecturer Erik Leander for valuable statistical advice     

Effect of the medicinal leech salivary gland secretion on the state of rat subcutaneous mast cells

It is firstly showed that the medicinal leech salivary gland secretion (SGS) as a polycomponent system of proteins and low-molecular weight substances, activates rat subcutaneous mast cells in vitro prompting a decrease in the heparin saturation index and increasing some characteristic mast cells morphometric parameters. The same mast cell changes were detected by analysis of some specimens of subcutaneous cellular tissue in the point of skin injured by the leech bite. It is shown that these changes are saved during 3 days. The mechanical injury of rat skin does not effect the mast cells activation. Activation of mast cells by SGS is extended to the distant subcutaneous mast cells. It is expressed in sharp decreasing of heparin saturation index although not statistically positive. The secondary leeching on these distant points provokes reduction of mast cells activation and some decrease of post-leeching blood heparin content: 0.154 +/- 0.03 units/ml (n = 10) as compared with post-leeching blood heparin contents analysed from the wound after the primary leeching (0.160 +/- 0.03 units/ml, n = 10). Proceeding from these findings, participation of heparin secreted from activated mast cells in the support of post-leeching bleeding is suggested, the phenomenon which provides unloading of capillary pool by application of medicinal leeches for treatment many diseases.     

Histidines are critical for heparin-dependent activation of mast cell tryptase

Mast cell tryptase is a tetrameric serine protease that is stored in complex with negatively charged heparin proteoglycans in the secretory granule. Tryptase has potent proinflammatory properties and has been implicated in diverse pathological conditions such as asthma and fibrosis. Previous studies have shown that tryptase binds tightly to heparin, and that heparin is required in the assembly of the tryptase tetramer as well as for stabilization of the active tetramer. Because the interaction of tryptase with heparin is optimal at acidic pH, we investigated in this study whether His residues are of importance for the heparin binding, tetramerization, and activation of the tryptase mouse mast cell protease 6. Molecular modeling of mouse mast cell protease 6 identified four His residues, H35, H106, H108, and H238, that are conserved among pH-dependent tryptases and are exposed on the molecular surface, and these four His residues were mutated to Ala. In addition, combinations of different mutations were prepared. Generally, the single His-Ala mutations did not cause any major defects in heparin binding, activation, or tetramerization, although some effect of the H106A mutation was observed. However, when several mutations were combined, large defects in all of these parameters were observed. Of the mutants, the triple mutant H106A/H108A/H238A was the most affected with an almost complete inability to bind to heparin and to form active tryptase tetramers. Taken together, this study shows that surface-exposed histidines mediate the interaction of mast cell tryptase with heparin and are of critical importance in the formation of active tryptase tetramers.     

Immunologic properties of mast cells from rats infected with Nippostrongylus brasiliensis.

The concentration of IgE in the serum of Sprague-Dawley rats increased after infection with Nippostrongylus brasiliensis (NB). The IgE concentration in normal rats was less than 1 mug/ml. After re-infection with NB, the concentration increased in 100 to 300 mug/ml. Mast cells were purified from peritoneal cells of both normal and NB-infected animals. Purified mast cells from the infected animals released histamine upon exposure to NB antigen. The antibody specific for IgE released histamine from purified mast cells of both normal and infected animals. Dose-reponse curves of histamine release suggested that mast cells from NB-infected animals bear more IgE molecules than normal mast cells. Binding of 125I-labeled rat E myeloma protein with normal mast cells was demonstrated by autoradiography. Under the same experimental conditions, mast cells of infected animals were not labeled with 125I-IgE. Mast cells from both normal and infected animals failed to combine 125I-labeled IgG. The number of IgE molecules bound per mast cell was determined by incubating 125I-labeled IgE with purified mast cells. When mast cells were incubated incubated in 0.6 to 2 mug/ml of IgE, the number of IgE molecules combined with the mast cells from infected animals was about 10% of that bound with normal mast cells. The results indicated that a large proportion of IgE receptors on mast cells of infected animals was occupied by their own IgE. No significant difference was observed between normal mast cells and those of infected animals with respect to histamine content and intracellular levels of cyclic nucleotides.     

Mast cells in the rat brain synthesize gonadotropin-releasing hormone

Mast cells occur in the brain and their number changes with reproductive status. While it has been suggested that brain mast cells contain the mammalian hypothalamic form of gonadotropin-releasing hormone (GnRH-I), it is not known whether mast cells synthesize GnRH-I de novo. In the present study, mast cells in the rat thalamus were immunoreactive to antisera generated against GnRH-I and the GnRH-I associated peptide (GAP); mast cell identity was confirmed by the presence of heparin, a molecule specific to mast cells, or serotonin. To test whether mast cells synthesize GnRH-I mRNA, in situ hybridization was performed using a GnRH-I cRNA probe, and the signal was identified as being within mast cells by the binding of avidin to heparin. GnRH-I mRNA was also found, using RT-PCR, in mast cells isolated from the peritoneal cavity. Given the function of GnRH-I in the regulation of reproduction, changes in the population of brain GnRH-I mast cells were investigated. While housing males with sexually receptive females for 2 h or 5 days resulted in a significant increase in the number of brain mast cells, the proportion of mast cells positive for GnRH-I was similar to that in males housed with a familiar male. These findings represent the first report showing that mast cells synthesize GnRH-I and that the mast cell increase seen in a reproductive context is the result of a parallel increase in GnRH-I positive and non-GnRH-I positive mast cells.     

The molecular role of mast cells in atherosclerotic cardiovascular disease

Human atherosclerosis has many characteristics of an inflammatory disorder. Recent data suggest that mast cells might be important in the pathogenesis of atherosclerotic disease. By secretion of pro-inflammatory cytokines, mast cells can assist in the recruitment of monocytes and lymphocytes into vascular tissue, thereby propagating the inflammatory response. Mast cell enzymes might activate pro-metalloproteinases, thereby destabilizing atheromatous plaques. Mast cells can facilitate foam cell formation by promoting cholesterol accumulation. However, mast cell tryptase could slow thrombus formation at sites of plaque rupture by interfering with coagulation. Therefore, mast cells can modulate coronary artery disease by both facilitatory and inhibitory pathways.     

Loss of histochemical identity in mast cells lacking carboxypeptidase A

Mast cell carboxypeptidase A (Mc-cpa) is a highly conserved secretory granule protease. The onset of expression in mast cell progenitors and lineage specificity suggest an important role for Mc-cpa in mast cells. To address the function of Mc-cpa, we generated Mc-cpa-null mice. Mc-cpa-/- mast cells lacked carboxypeptidase activity, revealing that Mc-cpa is a nonredundant enzyme. While Mc-cpa-/- peritoneal mast cells were ultrastructurally normal and synthesized normal amounts of heparin, they displayed striking histochemical and biochemical hallmarks of immature mast cells. Wild-type peritoneal mast cells had a mature phenotype characterized by differential histochemical staining with proteoglycan-reactive dyes (cells do not stain with alcian blue but stain with safranin and with berberine) and a high side scatter to forward scatter ratio by flow cytometry and were detergent resistant. In contrast, Mc-cpa-/- peritoneal mast cells, like immature bone marrow-derived cultured mast cells, stained with alcian blue normally or weakly and either did not stain with safranin and berberine or stained weakly, had a low side scatter to forward scatter ratio, and were detergent sensitive. This phenotype was partially ameliorated with age. Thus, histochemistry and flow cytometry, commonly used to measure mast cell maturation, deviated from morphology in Mc-cpa-/- mice. The Mc-cpa-/- mast cell phenotype was not associated with defects in degranulation in vitro or passive cutaneous anaphylaxis in vivo. Collectively, Mc-cpa plays a crucial role for the generation of phenotypically mature mast cells.     

Mast cell glycosaminoglycans

Mast cells contain granules packed with a mixture of proteins that are released on degranulation. The proteoglycan serglycin carries an array of glycosaminoglycan (GAG) side chains, sometimes heparin, sometimes chondroitin or dermatan sulphate. Tight packing of granule proteins is dependent on the presence of serglycin carrying these GAGs. The GAGs of mast cells were most intensively studied in the 1970s and 1980s, and though something is known about the fine structure of chondroitin sulphate and dermatan sulphate in mast cells, little is understood about the composition of the heparin/heparan sulphate chains. Recent emphasis on the analysis of mast cell heparin from different species and tissues, arising from the use of this GAG in medicine, lead to the question of whether variations within heparin structures between mast cell populations are as significant as variations in the mix of chondroitins and heparins.     

Degradation of the heparin matrix of mast cell granules by cultured fibroblasts

The ability of cultured rat fibroblasts to phagocytose rat peritoneal mast cell granules has been previously demonstrated by light and electron microscopy. To determine if the heparin matrix of ingested granules could be degraded by fibroblasts after phagocytosis, the heparin within peritoneal mast cells was labeled with [35S]sulfate in vivo. The 35S-labeled rat peritoneal mast cells were purified and their granules were isolated and shown to contain [35S]heparin proteoglycan. Incubation of [35S]heparin proteoglycan-containing granules with cultured rat fibroblasts revealed internalization of radioactivity by the fibroblasts over the first 24 hr consistent with phagocytosis of the granules by these fibroblasts. The [35S]heparin proteoglycan internalized by the fibroblasts was shown to decrease in size over 72 hr indicating that the fibroblasts were capable of degrading the heparin within the ingested granules. Degradation of [35S]heparin proteoglycan within the fibroblast was accompanied by the appearance of free [35S]sulfate in the extracellular compartment. Similar findings were obtained using cultured human fibroblasts. These data demonstrate for the first time that both rat and human fibroblasts are not only capable of ingesting mast cell granules but also of degrading mast cell granule heparin proteoglycan. This ingestion and degradation of mast cell granules by fibroblasts may represent an important mechanism in the regulation of the biologic expression of heparin and other granule-associated mediators in immediate hypersensitivity reactions.     

常用实验动物不同组织部位肥大细胞异质性的组织学特点比较

目的探讨并比较六种常用实验动物不同部位肥大细胞异质性的形态学特点。方法应用麻醉后处死的方法,取小鼠、大鼠、豚鼠、兔、犬和猴的皮肤、肺脏、乙状结肠和脾脏组织,经4%中性缓冲甲醛溶液或Bouin’s液固定后,制作常规组织切片,分别作HE、甲苯胺蓝、阿尔新蓝-藏红和P物质免疫组织化学染色,镜下观察并应用彩色病理图像分析软件进行形态学分析;另取皮肤组织固定于磷酸缓冲戊二醛溶液,制作常规超薄切片,透射电镜下观察肥大细胞超微结构。结果六种动物不同组织中肥大细胞各有其分布特点,且在形态大小、异染性及染色特性等方面表现各异,肥大细胞密度和形态学参数差异有显著性（P〈0.05）;豚鼠、犬和猴皮肤肥大细胞颗粒具有特殊亚微结构;小鼠皮肤组织内可见P物质免疫阳性肥大细胞和神经纤维,犬脾脏内可见P物质免疫阳性肥大细胞。结论在六种实验动物的同一组织中以及同一种动物不同组织中,肥大细胞具有明显的异质性,此异质性对采用实验动物开展与肥大细胞功能相关的动物实验研究具有重要参考价值。     

Changes in numbers and heparin content of peritoneal fluid mast cells of growing rats measured by flow cytofluorometry

A cytofluorometric method, based on berberine staining of mast cell heparin, was used for flow cytofluorometric counting and heparin quantitation of mast cells in crude peritoneal suspensions of growing rats. The automatic flow cytofluorometric counting of mast cells correlated well with hemocytometer cell counts. The mean mast cell heparin content obtained by flow cytofluorometry showed good agreement with such obtained by cytofluorometry of microscopically identified mast cells. The number of peritoneal mast cells and the mean mast cell heparin content was found to increase as the animals grew older. The results of the microscope fluorometric measurements suggested that the heparin content was normally distributed within mast cell populations of both young and old rats. However, the heparin distributions obtained by flow cytofluorometry were often positively skewed but did not fulfill the condition of the log-normal distribution.     

Mast cells exert effects outside the central nervous system to influence experimental allergic encephalomyelitis disease course

Previous studies using mast cell-deficient mice (W/W(v)) revealed that mast cells influence disease onset and severity of experimental allergic/autoimmune encephalomyelitis (EAE), the murine model for multiple sclerosis. The mast cell populations of these mice can be restored by transferring bone marrow-derived mast cells (BMMCs). Studies using the W/W(v) reconstitution model have lead to major advances in our understanding of mast cell roles in vivo. However, despite its common use, details regarding the sites and kinetics of mast cell repopulation have remained largely uncharacterized. In this study, we examined the kinetics and tissue distribution of green fluorescent protein(+) BMMCs in reconstituted W/W(v) mice to identify sites of mast cell influence in EAE. Reconstitution of naive animals with BMMCs does not restore mast cell populations to all organs, notably the brain, spinal cord, lymph nodes, and heart. Despite the absence of mast cells in the CNS, reconstituted mice exhibit an EAE disease course equivalent to that induced in wild-type mice. Mast cells are found adjacent to T cell-rich areas of the spleen and can migrate to the draining lymph node after disease induction. These data reveal that mast cells can act outside the CNS to influence EAE, perhaps by affecting the function of autoreactive lymphocytes.     

Mast cell products stimulate collagenase and prostaglandin E production by cultures of adherent rheumatoid synovial cells

Mast cells were purified from histologically-confirmed dog mastocytomas and extracted for whole mast cell products (MCP). When added to cultures of human adherent rheumatoid synovial cells MCP induced a 50-400 fold increase in prostaglandin E synthesis and a 10-50 fold stimulation of collagenase production. The mast cell stimulatory factor has not been identified and was not due to histamine, heparin or prostaglandin E. These results indicate a novel way in which mast cells might interact with synovial cells to promote the production of inflammatory mediators and proteolytic enzymes which might contribute to connective tissue degradation.