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.     

Demonstration of chymotryptic and tryptic activities in mast cells of rodents: comparison of 17 species of the family Muridae.

On the basis of studies in laboratory rats, mast cells were originally classified into two subgroups, namely, mucosal mast cells (MMCs), which contained chymase, and connective tissue mast cells (CTMCs), which contained both tryptase and chymase. This classification has been applied to other animal species, despite the fact that the MMCs and CTMCs of such species sometimes consist of mixed populations of mast cells in terms of tryptase and chymase constitution. This report describes the protease constitution of mast cells in 17 species of nine genera (Acomys, Apodemus, Cricetulus, Meriones, Millardia, Mus, Rattus, Sigmodon and Vandeleuria) of the family Muridae. MMCs with negative tryptase activity were detected only in the intestinal mucosa of six subspecies of Mus musculus, two Rattus spp. and Vandeleuria oleacea, and only Apodemus sylvaticus possessed CTMCs with no tryptase activity. Since mast cells conforming to the conventional classification were observed only in three of the nine genera examined, we propose that mast cells of rodents of the family Muridae should be classified by their protease constitution rather than by their location. Copyright Harcourt Publishers Ltd.     

Differential staining of mast cells with toluidine blue

Abstract

Mast cells play a significant role in inflammatory diseases such as asthma, inflammatory bowel disease, and autoimmune diseases. Inhibition of c-kit receptor tyrosine kinase, a growth factor receptor, significantly reduces mast cell numbers. The purpose of this study was to determine the effect of Compound X (a c-kit inhibitor) on mast cell numbers in rats. Connective tissue mast cells (CTMCs) and mucosal mast cells (MMCs) have differing histochemical characteristics which presents a challenge when staining for quantification by semi-automated image analysis. CTMCs are present in tissues such as tongue and skin and will stain readily in tissues fixed routinely. In contrast, MMCs, such as those present in the intestinal mucosa, are sensitive to fixation. Brief fixation in Carnoy’s solution, although seldom used due to its composition (a mixture of ethanol, chloroform, and acetic acid), was employed to fix tissues for MMC staining, while tissues for CTMC demonstration were fixed in 10% neutral buffered formalin. An enzyme histochemistry method, napthol AS-D choloroacetate (specific esterase), was briefly considered for staining; however, granulocytes stained along with mast cells, requiring manual identification and exclusion, thereby rendering the method incompatible with automated means of quantification. Instead, staining was performed using two different toluidine blue methods which have proven conducive to semi-automated image analysis techniques. CTMCs were stained using Luna’s toluidine blue, while MMCs were stained with Matsson’s toluidine blue modification. In summary, the selected methods, based upon a conventional stain, were easy to do and successfully identified both populations of mast cells for quantification by image analysis.     

Liver impairment after portacaval shunt in the rat: the loss of protective role of mast cells?

Mast cells are involved in various liver diseases and appear to play a broader pathogenic role than originally thought. They may participate in the splanchnic alterations related to a porto-systemic shunt. To verify this hypothesis we studied the serum and hepatic histological changes in rats four weeks after an end-to-side portacaval shunt. In this experimental model of chronic liver insufficiency we also assessed the mucosal mast cells (MMC) and connective tissue mast cells (CTMC) in the liver, mesenteric lymph nodes and small intestine, as well as the serum levels of rat mast cell protease-II (RMCP-II). The results show liver and testes atrophy, with hypoalbuminemia (p = 0.0001), hyperbilirubinemia (p = 0.0001) and increase in aspartate aminotransferase (p = 0.004) and alanine aminotransferase (p = 0.0001). Hepatic histopathology demonstrates hepatocytic necrosis and apoptosis, portal inflammation, biliary proliferation, steatosis and fibrosis. There is a decrease of MMCs and CTMCs in the liver, while in the ileum CTMCs increase and MMCs decrease. These results suggest the involvement of mast cells in the pathophysiological splanchnic impairments in this experimental model. In particular, the decreased number of liver mast cells may be associated with the hepatic atrophy. If this is the case, we propose that the disruption of the hepato-intestinal axis after a portocaval shunt in the rat could inhibit the ability of the liver to developing an appropriate repair response mediated by mast cells.     

Nerve growth factor treatment does not prevent dorsal root ganglion cell death induced by target removal in chick embryos

Summary In chick embryos, on the 3rd day of incubation, the developing right wing bud was removed. One group of the operated embryos was treated with a daily dose of 20 g purified nerve growth factor (NGF) from the 5th day of incubation and sacrificed on the 12th day. The other group was sacrificed on the 12th day of incubation and served as control. NGF was also administered to intact, unoperated embryos for comparison. The size of the dorsal root ganglia in segments 13–16 innervating the wings, were estimated and the number of surviving dorsal root ganglion cells counted both on the right (operated) and left (intact) sides. Although NGF brought about an increase in the size of the ganglia and an increase in the number of dorsal root ganglion cells bilaterally, it was not able to prevent excessive cell death of dorsal root ganglion cells on the operated side. The number of surviving neurons in the dorsal root ganglia on the operated side in embryos with or without NGF administration was only about 30–50% of the number of the intact side.These results show that cell death induced by target removal cannot be offset by NGF administration. It is concluded that NGF may act as a growth promoting agent for developing sensory neurons but other peripheral trophic factor/s are also needed for the maintenance and survival of dorsal root ganglion cells.     

Activation of peroxisome proliferator-activated receptor gamma suppresses mast cell maturation involved in allergic diseases

Background:  Mast cells play a central role in allergic and inflammatory diseases. Several reports indicated role of peroxisome proliferator-activated receptor gamma (PPARγ) on mast cell function. However, there is no report about the role of PPARγ on differentiation of mast cells from the progenitors. In this study, we investigated the role of PPARγ in regulating bone marrow-derived mast cell maturation and the therapeutic implications for mast cell-related diseases such as atopic or contact dermatitis.
Methods:  We used in vitro cell culture system for mast cell differentiation from bone marrow-progenitors using specific ligands and lentiviral-mediated short hairpin RNA of PPARγ, and in vivo murine dermatitis models.
Results:  Activation of PPARγ inhibited the maturation of bone marrow progenitors into connective tissue-type mast cells (CTMCs) through up-regulation of GATA-4 and GATA-6 resulting in a decrease in expression of histidine decarboxylase and mast cell histamine content. In comparison, the differentiation of bone marrow progenitors into CTMCs was significantly accelerated by the knockdown of PPARγ expression by lentiviral-mediated short hairpin RNA. Peroxisome proliferator-activated receptor gamma ligand administration to mice inhibited the maturation of mast cells resulting in attenuation of atopic and contact dermatitis via diminishment of the number of mature mast cells.
Conclusion:  Our results indicate that PPARγ is one of master regulators on mast cell maturation and potentially useful for the therapy in various disorders involving mast cell activation.     

The development of the trigeminal (V) motor nucleus in normal and tubocurare treated chick embryos

Summary The generation of cells and the naturally occurring neuronal death was studied in the trigeminal motor neuron pool in normal and tubocurare treated chick embryos between the 5th and 18th days of incubation. ³H-thymidine autoradiography revealed that the generation time extends from the 2nd to the 5th day of incubation, wherein about 50% of trigeminal motoneurons are born on the 3rd day. Maximum neuron number was found on the 7th day of incubation which steadily decreased to about 50% of the originally generated neurons by the 13th day. Nuclear pyknosis occurred from the 6th to the 13th day of incubation with a peak of neuron loss on the 7th day. Tubocurare, administered daily from the 5th day of incubation rescued most of the generated motoneurons which would otherwise have died. Cell nuclear area measurements in the motoneuron pool of the tubocurare treated animals showed a marked hypertrophy accompanying the increased neuronal survival.These observations indicate that tubocurare treatment prevents naturally occuring neuron death and causes significant nuclear hypertrophy within the trigemianal motoneuron pool innervating special, branchial arch derived muscles. Thus these neurons respond to tubocurare treatment in a manner similar to motoneurons of the spinal cord.     

Selective alterations in mast cell subsets and eosinophil infiltration in two complementary types of intestinal inflammation: ascariasis and Crohn's disease.

OBJECTIVE: Numbers of mast cells (MCs) of different subpopulations and the extent of eosinophil infiltration were compared in Crohn's disease and ascariasis. These two types of intestinal inflammation are complementary with regard to T cell response (TH1 versus TH2), prevalence and environmental factors. METHODS: Histochemical, immunohistochemical and ultrastructural tools were applied to biopsies of morphologically uninvolved colon, ileum and duodenum from Crohn's and ascariasis patients, as well as resection margins and tissues from an experimental porcine ascariasis model. MC subsets were defined by their dye-binding properties, and their chymase content was analysed using biochemical tools. RESULTS: The TH2 (IgE-mediated) response in ascariasis was characterised by a dramatic increase in mucosal- type MCs (MMCs) and eosinophils in both the mucosa and the deeper layers of the intestinal wall and a simultaneous decrease of connective tissue-type MCs (CTMCs). Uninvolved intestine of Crohn's patients showed moderate proliferation of CTMCs in the deeper layers of the intestinal wall, but a significant decrease of the MMCs, associated with moderate eosinophilia in all layers of the gut. Similar changes were present in the uninvolved duodenum of Crohn's patients. Comparable amounts of chymase could be extracted from mucosal and submucosal duodenum, with similar proportions of its two principal isoforms in each. CONCLUSIONS: Our results indicate that T cell responses (TH1 or TH2) are associated with different MC subsets in intestinal inflammation. Changes remote from the focus of inflammation point to the systemic nature of the different MC responses.     

The role of muscle activity in the differentiation of neuromuscular junctions in slow and fast chick muscles

Summary The differentiation of neuromuscular junctions of multiply innervated, slow, anterior latissimus dorsi (ALD) and focally innervated, fast, posterior latissimus dorsi (PLD) muscles was studied in normal and curarized chick embryos. At 16 days of incubation, fibres of both muscles are contacted by several axon profiles, the number of which falls with age. In 18-day-old embryos individual endplates in ALD are usually contacted by three axon profiles, whereas in PLD, endplates are contacted only by a single large terminal profile. At this time, there is already a significant accumulation of cell organelles in the postsynaptic area.Treatment of embryos with curare during the 7th and 12th day of incubation delays the differentiation of the neuromuscular junction in both muscles. The paralysis dramatically affects the decrease of the number of axon profiles at individual endplates in both muscles. At 16 days the number of axon profiles was greater in embryos treated with curare than in the untreated controls. At 18 days when the number of axon profiles normally decreases, the endplates of both types of curarized muscles have an even greater number of axon profiles than at 16 days. Endplates in curarized PLD had up to 13 and in curarized ALD up to 12 axon profiles. The effects of curare gradually wore off and when the movements of the embryos again became more vigorous, the normal differentiation of neuromuscular junctions continued. At 21 days of incubation many embryos recover from curare and show endplates of normal appearance in both muscles. These results suggest that activity of the muscle is essential for the maturation of the neuromuscular junctions.     

人类辅助生殖技术中DAY3胚胎质量与囊胚形成相关性分析

目的探讨人类辅助生殖技术中,day3胚胎质量与囊胚形成的相关性。方法将符合纳入标准的130例患者day3胚胎移植后剩余胚胎进行囊胚培养至day6,分析患者年龄、day3胚胎分级、day3胚胎细胞数及培养时间与囊胚形成的关系。结果患者年龄与囊胚形成无明显相关;囊胚形成率与day3胚胎质量评分和细胞数呈显著正相关,day3评分为Ⅰ-Ⅱ级胚胎囊胚形成率明显高于Ⅲ-Ⅳ级胚胎,day3细胞数≥6的胚胎囊胚形成率明显高于≤5的胚胎囊胚形成率。综合分析day3胚胎分级、细胞数、培养时间与囊胚形成率的关系,囊胚形成率最高的是≥6细胞Ⅰ-Ⅱ级胚胎,其次是≥6细胞Ⅲ-Ⅳ级胚胎,≤5细胞的其他两组囊胚形成很低,各组胚胎day6囊胚形成率均明显高于day5。结论 day3胚胎的细胞数、质量分级及囊胚的培养天数都能够影响囊胚的形成,其中day3胚胎的细胞数更为重要,培养至day6使day3胚胎质量较差和发育迟缓的胚胎有机会形成囊胚,有效地筛选和保存了有一定发育潜能的胚胎。     

Macrophages during avian optic nerve development: relationship to cell death and differentiation into microglia

Cell death is frequent during the development of the nervous system. In the developing optic nerve of chicks and quails, neuroepithelial cell death was first observable on the third day of incubation, slightly after the first cell ganglion axons appeared in the stalk. Specialized phagocytes were observed within the stalk in chronological and topographical coincidence with cell death. These cells were identified as macrophages because of their morphological features, intense acid phosphatase activity and, in quail embryos, labeling with QH1, a monoclonal antibody recognizing quail hemangioblastic cells. Macrophages in areas of cell death were round and actively phagocytosed cell debris. We used electron microscopy and histochemical and immunocytochemical labeling to study macrophagic cells of the optic nerve in avian embryos of 3–6.5 days of incubation. As development proceeded, phagocytosing, round macrophages became ameboid macrophages that migrated from areas of cell death toward regions occupied by optic axonal fascicles. Macrophages in these locations were thin and elongated, with a few processes. To elucidate the final fate of macrophagic cells in the optic nerve, sections taken from older embryonic and hatched quails were stained with the QH1 antibody. On the 8th day of incubation some slightly ramified QH1+ cells were present among axonal fascicles. In subsequent stages these cells increased in number and acquired more complex ramifications. In adult optic nerves, QH1+ cells had a small body and sent out slender processes, sometimes with secondary and tertiary branches, which were frequently orientated parallel to the course of the optic axons. These cells were considered to be microglial cells. The appearance of macrophages within the developing optic nerve at the same time as neuroepithelial cell death suggests that cell death influences the recruitment of macrophages into the nerve. When macrophages reach the areas invaded by optic axonal fascicles, they undergo structural and probably also physiological changes that appear to signal differentiation into microglia.     

IL-18 induces a marked gene expression profile change and increased Ccl1 (I-309) production in mouse mucosal mast cell homologs

Helminthic infections, which are particularly common in the developing world, are associated with the accumulation of mucosal mast cells (MMCs) in the epithelial layer of the gut. Although intestinal parasite infection models argue that IL-18 plays a role in MMC differentiation and function, the direct effect of IL-18 on MMCs is still not well understood. To clarify the role of IL-18 in mast cell biology, we analyzed gene expression changes in MMCs in vitro. DNA microarray technology uncovered a group of chemokines regulated by IL-18, among which Ccl1 (I-309, TCA-3) showed the highest up-regulation. Ccl1 induction was only transient in mast cells and was characteristic for both immature and mature MMCs, but not for connective tissue-type mast cells. IL-18 exerts its Ccl1-inducing effect in MMCs primarily via the activation of NFkappaB. Moreover, IL-18 was effective both in the absence and the presence of IgE-antigen complex. The Ccl1 receptor (CCR8) is known to be expressed by T(h)2 cells and is involved in their recruitment. Our present findings suggest that IL-18 may contribute to mast cell-influenced Th2 responses by inducing Ccl1 production.     

The development of the chick embryo gallbladder studied by scanning electron microscope

The differentiation of the gallbladder mucous membrane in chick embryos has been studied from the 11th day of incubation until hatching (stages 37-46 of Hamburger an Hamilton 1951) with scanning electron microscope. The surface of the mucous membrane, at the 11th day of incubation, appears regularly smooth. From the 12th day onwards, longitudinal folds appear on the surface of the mucous membrane, becoming more and more numerous and complicated in the following days. During the last days of incubation, branching and anastomosing folds are present. At the 11th day of incubation, the epithelial cells lining the gallbladder surface have flattened apices, with short microvilli. From the 12th day onwards, the epithelial cells show dome shaped apices, with long numerous microvilli. From the 15th day of incubation onwards, some particular secretory cells can be detected. The appearance of these cells is probably related to the water-absorbing function of the gallbladder which in the last days of incubation completes its functional development, because of precise digestive requirements of the chick embryo.     

Murine interleukin-2 generates glycogen-rich and mucus-secreting NK cells.

Colonies of cells termed 'giant granular leucocytes' (GGL) displaying natural killer (NK) activity were generated in cell culture. The prominent feature of these cells was the formation of large cytoplasmic pool--the 'theca'--filled up with glycogen. This was demonstrated by the strong positive red staining of the theca with periodic acid Schiff reagent (PAS) which was abolished by prior treatment with amylase. Two different procedures were employed for obtaining colonies of NK-GGL. In the first, mice were injected either with killed Corynebacterium parvum or with killed Bordetella pertusis preparations and their mesenteric lymph-node cells were grown on syngeneic X-irradiated embryonic skin fibroblast monolayers. At the foci of GGL formation the fibroblasts were killed and the cleared areas thus formed were populated by adherent GGL. In the second procedure, supernates from rat or mouse spleen cultures stimulated with concanavalin A (Con A)--Interleukin-2 (IL-2)--were added to cultures of spleen and lymph-node cells prepared from either ordinary or from athymic nude mice. Richest GGL populations developed when rat IL-2 was added to cells of nude mice. Mouse IL-2 was less consistent. With nude mouse cells it stimulated, either mast cells or GGL, or both; rat IL-2 did not stimulate mast-cell differentiation in nude mouse cultures. In contrast, supernates from lymph-node cell cultures prepared from mice infected with Schistosoma mansoni. Mucosal mast cell-stimulating factor (MMSF) stimulated the formation of colonies of mast cells but not GGL. When MMSF was added as late as 23 days, colonies of young mast cells appeared and mast cells progressively increased in number. When rat IL-2 was added to such mature mast-cell cultures on the 30th day, colonies of cytolytic-GGL appeared. These observations indicate that precursors of mast cells and GGL persist in the cultures and preserve their potential to be stimulated by T-cell factors. GGL-NK cells developed on monolayers prepared from whole embryos released substance that displayed morphology and staining characteristic of mucus. Evidence gathered from in-vitro and in-vivo studies links the in-vitro GGL-NK cells to motile cells that inhabit the mucosal epithelium. Based on the observations, a hypothesis on the function of NK cytotoxicity is brought forward. It proposes the replacement of ordinary epithelial cells, which are killed during a proliferative and differentiative response of other cells at the onset of an infection course.     

Effect of coculture of rodent mast cells with murine chronic graft-versus-host disease (cGVHD)-derived fibroblasts.

We investigated whether murine chronic graft-versus-host disease (cGVHD)-derived skin fibroblasts maintain the viability and functional activity of rat peritoneal connective tissue-mast cells (CTMCs) and whether they affect the change in phenotype of mouse bone marrow-derived mast cells (BMMCs). Skin fibroblasts were isolated before the development of fibrosis (day 5), during overt fibrosis (day 28), and after recovery from fibrosis (day 120). cGVHD fibroblasts of days 5 and 28 exhibited enhanced proliferation, a property that was maintained through several subcultures. CTMCs adhered to the same extent, did not divide, and maintained their viability in all the different cultures. When CTMCs were activated with compound 48/80, they released approximately 80% of their histamine content, indicating that coculture with cGVHD fibroblasts did not adversely affect CTMC function. The amount of histamine found in the medium of 8 days CTMC/cGVHD fibroblast coculture was similar to that found in control culture. These findings suggest that the degranulation of dermal MCs, characteristic of cGVHD, is not due to a direct activating effect of the cGVHD fibroblasts on the MCs. BMMCs seeded on cGVHD fibroblasts acquired the capacity for safranin staining and increased their histamine content, indicative of a change to CTMCs. Thus, cGVHD fibroblasts are able to provide a microenvironment adequate for maintaining viability and activity of CTMCs and for promoting maturation and change in phenotype of BMMCs.     

Natural infection of duck embryos with duck hepatitis B virus: time and tissue sequence of infection

There have been no studies addressing the detailed sequence of embryonic infection with duck hepatitis B virus (DHBV). Therefore, duck embryos from flocks infected with DHBV were examined to study the sequence of infection by DHBV in various embryonic tissues. Embryos from flocks infected with DHBV were harvested in duplicates from 7 to 25 days of incubation. Whole embryos (to 12 days) or dissected embryonic tissues were fixed, paraffin embedded, and stained for DHBV surface antigen (DHBsAg) using a peroxidase-antiperoxidase technique. Isolated hepatic cells were infected in 7-day-old embryos, and these increased in number until 11 days, when most cells were positive for DHBsAg. Endocrine pancreatic cells were positive from day 10, but only an occasional exocrine pancreatic cell was infected after day 20. Renal tubule cells were positive for DHBV by day 11, increasing in number until about day 18, after which a decline in numbers of infected cells occurred. Renal glomeruli became positive for DHBsAg from day 24. When present in the developing embryo, thymus, bursa of Fabricius, spleen, bone marrow, lung, and duodenum remained negative for DHBsAg. It was concluded that the timing of infection of specific tissues was not necessarily related to cellular maturity but may reflect a need for specific metabolic functions that permit viral replication.     

扬子鳄胚胎舌表面的扫描电镜观察

在扫描电镜下观察了孵化第３６ｄ至第６２ｄ孵出的扬子鳄（Ａｌｌｉｇａｔｏｒｓｉｎｅｎｓｉｓ）胚胎舌表面的形态变化过程。舌表面于孵化第３８ｄ形成少数大的隆起与凹陷，第４刚开始出现舌乳头。舌上皮细胞的表面在第３６～４２ｄ为圆形，表面光滑，中央凹陷，以后逐渐变成扁平细胞，第５２ｄ细胞呈规则的多边形，表面微绒毛清晰，细胞中央有一向外隆起的圆形或卵圆形核区，第５６ｄ后老化的舌上皮出现脱落现象。孵化第４２ｄ舌后部及中部的上皮内陷形成较大的舌腺腺孔。第４８～５６ｄ中，较小的舌腺孔显著增多，而大舌腺孔数目无明显增加。第６２ｄ多数大舌腺孔内可见有粘液样分泌物。舌表面味蕾的形成很迟，第５６ｄ才出现Ⅰ、Ⅱ、Ⅲ型发育中的味蕾，第６２ｄ形成部分成熟的味蕾。本文对扬子鳄舌腺及味蕾的形态发生特点作了讨论。     

Expression of integrin-alphaE by mucosal mast cells in the intestinal epithelium and its absence in nematode-infected mice lacking the transforming growth factor-beta1-activating integrin alphavbeta6

Peak intestinal mucosal mast cell (MMC) recruitment coincides with expulsion of Trichinella spiralis, at a time when the majority of the MMCs are located within the epithelium in BALB/c mice. Although expression of integrin-alpha(E)beta(7) by MMCs has not been formally demonstrated, it has been proposed as a potential mechanism to account for the predominantly intraepithelial location of MMCs during nematode infection. Co-expression of integrin-alpha(E)beta(7) and the MMC chymase mouse mast cell protease-1, by mouse bone marrow-derived mast cells, is strictly regulated by transforming growth factor (TGF)-beta(1). However, TGF-beta(1) is secreted as part of a latent complex in vivo and subsequent extracellular modification is required to render it biologically active. We now show, for the first time, that intraepithelial MMCs express integrin-alpha(E)beta(7) in Trichinella-infected BALB/c and S129 mice. In S129 mice that lack the gene for the integrin-beta(6) subunit and, as consequence, do not express the epithelial integrin-alpha(v)beta(6), integrin-alpha(E) expression is virtually abolished and recruitment of MMCs into the intestinal epithelium is dramatically reduced despite significant overall augmentation of the MMC population. Because a major function of integrin-alpha(v)beta(6) is to activate latent TGF-beta(1,) these findings strongly support a role for TGF-beta(1) in both the recruitment and differentiation of murine MMCs during nematode infection.