Toluidine Blue-positive Mast Cells Research Articles

Mast cell distribution in porcine common bile duct with special reference to ghrelin

Due to the similar pattern of ghrelin localisation in human and porcine intestine, swine are described as a suitable animal model for experiments related to ghrelin and its receptors. In a previous study of ours, the presence of ghrelin immunoreactive endocrine cells in common bile duct (Ductus choledochus) (CBD) of domestic swine was established. However, data regarding the distribution of mast cell types in the CBD in this species are missing. The current study aimed to determine the distribution of tryptase-, ghrelin- and toluidine blue positive mast cells in the wall of extra- and intramural parts of porcine CBD. For this purpose, immunohistochemical staining for detection of tryptase was performed in order to identify ghrelin reactivity in mast cells. Additionally, toluidine blue staining was carried out to compare the number of metachromatic mast cells with that of tryptase positive cells. The comparative quantitative analysis showed that the number of tryptase immunorective cells was higher than that of metachromatic ones in the CBD wall. Ghrelin immunoreactive cells were the most numerous, therefore it can be assumed that not only mast cells but other cells also contain ghrelin. The three mast cells types were more abundant in the propria and subserosa of the intramural part than in the same layers of extramural CBD part. In the muscle layer, mast cell number was higher in the extramural than in the intramural part of the duct. In conclusion, taking into account the results obtained, an important role of mast cells play in controlling CBD motility and biliary gland function was suggested.

Mast cell density in domestic swine common hepatic duct

Detailed mast cell classification is reported in humans and rats, however such classification is not available in porcine common hepatic duct. It is interesting to find out whether mast cells in common hepatic duct are able to produce ghrelin, which is involved in a series of biological functions including regulation of food intake, body weight, gastrointestinal motility, hormone secretion, glucose release, cardiovascular functions, enzyme release, cell proliferation and reproduction in pigs. Because the determination of the cellular mechanisms responsible for normal and pathological biliary tract motility is difficult in humans, swine appears to be one of the most suitable animal model for physiological and morphological studies related to ghrelin peptide. The lack of information about the distribution of mast cells in the wall of common hepatic duct (Ductus hepaticus communis) in domestic swine motivated us to undertake this study in order to gain a better understanding of ghrelin peptide. The aim of this study was to define the localisation and number of tryptase-, ghrelin- and toluidine blue positive mast cells in the layers of porcine common hepatic duct. In this respect, the immunohistochemical staining for detection of tryptase and ghrelin expression was performed. The toluidine blue staining allowed the visualisation of metachromatic mast cells. The comparative study showed that tryptase positive cells were more numerous than metachromatic cells in all layers of the studied organ but the density of ghrelin positive cells was the highest. Tryptase immunohistochemistry allowed distinguishing ghrelin expressing mast cells from all ghrelin positive cells. The highest number of ghrelin positive cells indicated that not only mast cells but other cells also express ghrelin. The results allowed concluding that studied mast cell types had different density in the three main layers of common hepatic duct. The highest density of mast cells in the muscular layer and the possibility of mast cells to express ghrelin define its role in regulation of common hepatic duct motility and glandular secretion.

C1Q‐TNF‐related peptide 8 (CTRP8) in human prostate cancer

Introduction C1Q Tumor Necrosis Factor related Peptide 8 (CTRP8) is the one of the least characterized members of the C1Q/TNF family of proteins which is engaged in diverse functions ranging from metabolism to immunity and is one of only two CTRP family members absent in the mouse genome. CTRP8 was identified in our lab as a novel ligand of the RXFP1 receptor and was found to promote invasiveness and chemoresistance to temozolomide in glioblastoma (GB)(Glogowska et al., 2013; Thanasupawat et al., 2018). We have generated high-affinity rabbit antisera to human CTRP8 to identify a selective mast cell subpopulation in human prostate cancer tissues (Hempel Sullivan et al., 2020). Mast cells are innate immune cells which are primarily known for their role in the mediation of allergic responses. They have been detected within prostate cancer tissues and serve as potential prognostic marker in prostate cancer (Johansson et al., 2010; Hempel Sullivan et al., 2020). Methods Affinity-purified rabbit polyclonal antisera directed against an epitope unique to human CTRP8 were characterized by Western blot, immunofluorescence, and immunohistochemistry in prostate cancer tissue sections and human prostate cancer tissue microarrays (TMAs). Results Of several Flag-tagged human and mouse CTRP family members transiently expressed in HEK293T cells, the CTRP8 antisera exclusively detected human CTRP8 as assessed by Western blot, demonstrating the specificity of the antisera. We studied the expression of CTRP8 in human prostate tissues. CTRP8 was expressed in a subpopulation of human mast cells distributed in prostate cancer tissues, as demonstrated by co-localization of CTRP8 immunoreactivity and toluidine blue positive mast cells in prostate cancer tissues. This was further validated by dual immunofluorescence of CTRP8 with Tryptase, a marker for mature mast cells. Initial analysis of prostate cancer tissue microarrays (TMAs) (n= 360) revealed a comparative increase in the proportion of Tryptase+/ CTRP8+ cells versus total number of mast cells (Tryptase+/ CTRP8+ and Tryptase+/ CTRP8- cells) in the extra-tumoral compartment as compared to intra-tumoral regions for Gleason grades 6 and 7 prostate cancer samples. Weak staining of CTRP8 was detected in prostate epithelial cells. The RXFP1+ human prostate cancer cell line PC3 responded to CTRP8 treatment with increased protein kinase C delta signaling and enhanced motility as determined by real-time migration assays. Conclusions Here we have identified the expression of adipokine family member CTRP8 in prostate epithelial cells and in a subpopulation of mature mast cells in patient prostate cancer tissues, thus, identifying CTRP8 as a novel player in prostate cancer and associated mast cell compartment. References Glogowska, A., et al. J. Pathol., 231, 466–479, 2013 Thanasupawat, T., et al. Mol. Oncol. 12, 1464–1479, 2018 Johansson, A., Am. J. Pathol, 177, 1031–1041, 2010 Hempel Sullivan H, et al. J. Pathol., 2020 Hempel Sullivan H, et al. Cancer Epidemiol Biomarkers Prev., 2020

Comparative study of heparin- and toluidine blue positive mast cells in porcine lumbar spinal ga

Comparative study of heparin- and toluidine blue positive mast cells in porcine lumbar spinal ga

Distribution of ghrelin-positive mast cells in rat stomach

ABSTRACTIt is known that the gastrointestinal peptide hormone ghrelin is expressed in human and rodent B lymphocytes, T lymphocytes, monocytes and natural killer cells. However, there are no data about ghrelin expression by mast cells. These facts, as well as the common progenitor cells of mast cells and the above-mentioned immune cells, motivated us to undertake the current work in order to prove that like other granulocytes, rat gastric mast cells are capable of immunohistochemical expression of ghrelin. Gastric wall sections of Wistar rats were studied immunohistochemically for detection of ghrelin and tryptase and histochemically for toluidine blue in order to identify ghrelin-positive mast cells as well as to establish their localization and distribution. Results showed that mast cell granules expressed ghrelin. The ghrelin-positive mast cells were the least numerous as compared to tryptase-positive mast cells and toluidine blue-positive mast cells. Based on the observed expression of ghrelin in granules of mast cells localized in the rat gastric wall, we suggested that this type of cell can be regarded as an important source of ghrelin and suggested that ghrelin may exert different physiological functions, such as regulation of muscular, epithelial and glandular functions.

Involvement of Mast Cells in α7 Nicotinic Receptor Agonist Exacerbation of Freund's Complete Adjuvant-Induced Monoarthritis in Mice.

Activation of antiinflammatory cholinergic (vagal) pathways can reduce inflammation, and in vitro studies support a pivotal role of α7 nicotinic acetylcholine receptors (α7-nAChR), macrophages, and T cells in these events. The aim of this study was to assess α7-nAChR agonists as an antiinflammatory treatment for Freund's complete adjuvant (CFA)-induced monoarthritis. Arthritis was induced by intraarticular injection of CFA unilaterally into the knee joints of mice. Animals were treated with α7-nAChR agonists (AR-R17779 or A844606), with or without antagonists (COG133 or methyllycaconitine), and joint inflammation and pain were assessed. Experiments were repeated in c-Kit(W-sh) mast cell-deficient mice, and the effects of an α7-nAChR agonist on mast cell proliferation, migration, and activation by lipopolysaccharide (LPS) were tested. Treatment with α7-nAChR agonists significantly exacerbated CFA-induced arthritis and pain, as gauged by all indices of assessment, the specificity of which was confirmed by coadministration of an nAChR antagonist that attenuated the increase in disease severity. Toluidine blue-positive mast cells were increased in the joint capsule of CFA plus AR-R17779-treated mice, and AR-R17779 enhanced LPS-induced TNF proliferation and migration of a human mast cell line. The AR-R17779-driven increase in severity of CFA-induced arthritis was significantly reduced in mast cell-deficient mice. Using CFA to elicit a local inflammatory response, we found that pharmacologic activation of α7-nAChR exacerbated joint inflammation and pain, in part via mast cells, which illustrates the organ- and disease-specific nature of regulatory neuroimmune mechanisms. Thus, α7-nAChR activation may not be uniformly antiinflammatory in all types of inflammatory joint disease.

Histochemical Study of Heparin-positive Mast Cells in the Terminal Part of Porcine Ductus Choledochus and Papilla Duodeni Major.

The study presented in detail the localization and density of mast cells (MCs) in the intramural part of the common bile duct (CBD) and in the major duodenal papilla (MDP) of domestic swine. MCs' density (number/mm(2) ) in different layers of both of the duct and papilla was evaluated after toluidine blue staining. Their number was higher in the lamina propria mucosae than in the tunica muscularis of the studied structures. The localization of berberine-positive, (heparin containing) MCs and the ratio between them and toluidine blue-positive MCs with γ-ma metachromasia was also established. Ratios of heparin-containing MCs in comparison with all toluidine blue-positive MCs were found as follows: ductus choledochus - 32% in the subglandular connective tissue of lamina propria mucosae in the intramural part of the duct; m. sphincter ductus choledochus - 31% in the circular and 0.06% in the longitudinal muscle layer; subserosa - 59%; papilla duodeni major - 0.03% in the subepithelial connective tissue and 34% in the subglandular connective tissue of lamina propria mucosae, respectively. The established large difference in heparin-positive MCs in both the subepithelial and subglandular connective tissues of CBD and MDP, respectively, is an evidence for the existence of mucosal and connective tissue MCs.

Urinary and faecal N-methylhistamine concentrations do not serve as markers for mast cell activation or clinical disease activity in dogs with chronic enteropathies.

BackgroundThis study sought to correlate faecal and urinary N-methylhistamine (NMH) concentrations with resting versus degranulated duodenal mast cell numbers in dogs with chronic enteropathies (CE), and investigate correlations between intestinal mast cell activation and clinical severity of disease as assessed by canine chronic enteropathy clinical activity index (CCECAI), and between urinary and faecal NMH concentrations, mast cell numbers, and histopathological scores. Twenty-eight dogs with CE were included. Duodenal biopsies were stained with haematoxylin and eosin (H&E), toluidine blue, and by immunohistochemical labelling for tryptase. Duodenal biopsies were assigned a histopathological severity score, and duodenal mast cell numbers were counted in five high-power fields after metachromatic and immunohistochemical staining. Faecal and urinary NMH concentrations were measured by gas chromatography–mass spectrometry.ResultsThere was no correlation between the CCECAI and faecal or urinary NMH concentrations, mast cell numbers, or histopathological score – or between faecal or urinary NMH concentration and mast cell numbers. Post hoc analysis revealed a statistically significant difference in toluidine blue positive mast cells between two treatment groups (exclusion diet with/without metronidazole versus immunosuppression (IS)), with higher numbers among dogs not requiring IS.ConclusionFaecal and urinary NMH concentrations and duodenal mast cell numbers were not useful indicators of severity of disease as assessed by the CCECAI or histological evaluation. The number of duodenal mast cells was higher in dogs that did not need IS, i.e. in dogs responding to an exclusion diet (with/without metronidazole), than in dogs requiring IS. Further studies comparing the role of mast cells in dogs with different forms of CE are needed.

Galectin-3 Gene Inactivation Reduces Atherosclerotic Lesions and Adventitial Inflammation in ApoE-Deficient Mice

This study has examined the role of galectin-3 (GaL3), a multicompartmented N-acetyllactosamine-binding chimeric lectin, on atherogenesis in the ApoE-deficient mouse model of atherosclerosis. Pathological changes consisting of atheromatous plaques, atherosclerotic microaneurysms extending into periaortic vascular channels, and adventitial and periaortic inflammatory infiltrates were assessed in an equal number (n = 36) of apolipoprotein (Apo)E-deficient mice and ApoE-GaL3 double-knockout mice. These mice were divided into three age groups, 21 to 23 weeks, 25 to 31 weeks, and 36 to 44 weeks of age. Results of this morphological analysis have shown an age-related increase in the incidence of aorta atheromatous plaques and periaortic vascular channels in ApoE-deficient mice. By contrast ApoE/GaL3 double-knockout mice did not show an increase in pathological changes with age. The 36- to 44-week group of ApoE(-/-)/GaL3(-/-) mice had a significantly lower number of atherosclerotic lesions (P < 0.004) and fewer atheromatous plaques (P < 0.008) when compared with ApoE(-/-)/GaL3+/+ mice of the same age. ApoE(-/-)/GaL3(-/-) mice had a lower number of perivascular inflammatory infiltrates and mast cells than those found in ApoE(-/-)/GaL3+/+ mice. The reduced number of perivascular mast cells may have resulted in a low level of interleukin-4 that contributed to the reduction in the morphological parameters of atherogenesis correlated with the lack of GaL3 expression. The effect of GaL3 deficiency on atherogenesis decrease could be related to its function as a multifunctional protein implicated in macrophage chemotaxis, angiogenesis, lipid loading, and inflammation.

Identification and localization of adrenomedullin-storing cardiac mast cells

Adrenomedullin (AM), a potent vasodilatory hypotensive peptide, is expressed in the heart, where it is known to play a protective action. Light-microscopy immunocytochemistry (ICC) demonstrated the presence of AM immunoreactivity not only in the coronary-vessel wall and ventricular myocytes of the human and rat heart, but also in sparse voluminous cells located in the perivascular space. These cells displayed the same location of toluidine blue-positive mast cells, and electron microscopy ICC showed AM-immunogold staining over the granules of rat cardiac mast cells. The incubation of rat left ventricle fragments with the mast-cell histamine releaser compound 48/80 evidenced groups of AM-positive cells undergoing degranulation and caused an increase of approximately 50% in the AM concentration in the incubation medium. Collectively, our findings provide evidence that at least a subset of cardiac mast cells are able to synthesize and store AM, and upon stimulation to release it near coronary arterioles and venules.

Adrenomedullin, ANP and BNP are colocalized in a subset of endocrine cells in the rat heart

We investigated by immunocytochemistry (ICC) the distribution in the rat heart of adrenomedullin (AM), a potent and long-lasting hypotensive peptide which is expressed in the cardiovascular system, where it is known to play a major regulatory role. Hearts were collected from adult male Sprague-Dawley rats, and were perfused for 20 min, according to the Langendorff technique, with endothelin-1 (ET-1) or the mast cell-degranulator compound 48/80. Hearts were frozen, and ICC was performed using standard techniques and a specific anti-rat AM1-50 antibody. We confirmed the presence of a low AM-immunoreactivity in cardiomyocytes and cardiac fibroblasts, as well as in endothelial and smooth muscle cells of coronary vessels. Moreover, we provided evidence of the presence in both atria and ventricles of sparse voluminous AM-positive cells, mainly located near coronary vessels. These cells had the same juxtavascular location of toluidine blue-positive mast cells and their number decreased upon acute exposure to the 48/80 compound. However, ICC showed that in these cells AM was always colocalized with atrial and brain natriuretic peptides. Moreover, AM-storing cells were also positive to MyHC-Apla2, indicating that they share some phenotypic features with immature smooth muscle cells. The number of AM-storing cells underwent a dramatic decrease in response to the potent vasoconstrictor ET-1, suggesting an acute release of stored vasodilatory AM aimed at counteracting coronary constriction. Taken together, our present findings support the hypothesis that these cells may represent a novel subset of endocrine cells, strategically located near blood vessels in the mammalian heart, where they can release vasoactive peptides.

Effect of granulocyte macrophage colony-stimulating factor in a patient with benign systemic mastocytosis.

We report the in vitro and in vivo effects of granulocyte macrophage colony stimulating factor (GM-CSF), a known inhibitor of in vitro mast cell differentiation, in a patient with benign, adult-onset systemic mastocytosis. In vitro effects of GM-CSF on bone marrow cultures before the start of treatment showed a marked inhibition of mast cell marker expression [tryptase, Kit, and high-affinity IgE receptor (FcepsilonRIalpha)] at both protein and mRNA levels. Therefore, the patient was treated with daily injections of GM-CSF for 10 weeks. After an initial improvement, increasing worsening of clinical symptoms was noted, and the patient refused further treatment. Lesional skin biopsies showed an increase of toluidine blue-positive mast cells, compared with uninvolved skin, with further significant increase after treatment. Similar results were obtained on staining for mast cell-specific tryptase and Kit, as well as for CD1a and FcepsilonRIalpha. These findings show that GM-CSF inhibits human bone marrow mast cell differentiation in vitro, and also in mastocytosis. However, GM-CSF apparently enhances recruitment of mast cell as well as dendritic cell precursors into the tissue during systemic treatment. These findings and the observed adverse clinical effects in the present patient make it unlikely that GM-CSF monotherapy will be beneficial for the treatment of mastocytosis.

Enzyme-histochemical Detection of a Chymase-like Proteinase within Bovine Mucosal and Connective Tissue Mast Cells

The presence of chymase-like proteinase in bovine mast cells was investigated by an enzyme-histochemical technique (naphthol-AS-D-chloroacetate as substrate) in normal skin, primary bronchus, lung and duodenum. The counts and distribution of chymase-positive and toluidine blue-positive mast cells were compared by means of successive staining. Mast cells with chymase-like activity were detected in all areas, but their proportion was greater in connective than mucosal tissues, with the exception of the skin. These results contrast with those obtained in rodents, in which chymase-like proteinases are detected in all tissues and also in all mast cells. Bovine mast cells are closer to those of human beings, in which chymase-containing mast cells predominate in connective tissues, including skin. The results suggest that more than one chymase subset is present, at least in duodenum. The possible occurrence of dual-specific chymase mast cells, as in other ruminants, is discussed.

Brain mast cell degranulation regulates blood-brain barrier.

Mast cells synthesize vasoactive agents and a number of neurotransmitters. They are particularly numerous in the medial habenular region of the epithalamus, the attachment site of the choroid plexus. The present study examined whether degranulation of brain mast cells alters the permeability of the blood-brain barrier (BBB). To this end, doves were injected intramuscularly with the mast cell degranulator, compound 48/80 (C40/80), followed by i.v. injection of Evans blue. The distribution of the dye in the parenchyma was examined using digital imaging. Three brain areas were analyzed: the medial habenula (which also contains mast cells), the paraventricular nucleus (PVN, which abuts the third ventricle, but has no mast cells), and the lateral septal organ (LSO, a circumventricular organ with fenestrated capillaries). Significantly more Evans blue tracer and fewer toluidine blue-positive mast cells were detected in the medial habenula of subjects treated with C48/80 compared to saline controls. Evans blue did not enter the PVN in either the experimental or control group, while it entered the LSO equally in both. Degranulation of mast cells after C48/80 treatment was confirmed histochemically and ultrastructurally. The results support the hypothesis that brain mast cell degranulation locally alters BBB permeability. Activation of brain mast cells may provide a mechanism for regulated opening of the BBB.

Expression of stem cell factor in cutaneous mastocytosis

Stem cell factor has recently been identified as a potent growth factor for bone marrow stem cells, melanocytes and mast cells. In order to evaluate its possible role in human mastocytosis, skin lesions from 13 patients with urticaria pigmentosa and five patients with mastocytomas, and normal skin specimens from five healthy donors were studied by immunohistochemistry, using polyclonal and monoclonal (hkl-12) antibodies against stem cell factor, and a monoclonal antibody (YB5.B8) against its receptor, the c-kit proto-oncogene product. Stem cell factor expression was noted in all sections studied, with an equal distribution pattern for both antibodies, but a weaker intensity with the hkl-12 reagent. Cytoplasmic staining was noted in keratinocytes, Langerhans cells, sweat gland ductal lining cells, mast cells, endothelial cells and spindle-shaped dermal stromal cells. An intense, diffusely granular reaction pattern was noted in all cells, except for a sparse, coarsely granular pattern in mast cells and stromal cells. In urticaria pigmentosa, staining was weaker in keratinocytes, but more prominent in Langerhans cells. In all sections, toluidine blue-positive mast cells and TA 99-positive basal epidermal melanocytes were the only cells to react with the c-kit antibody. Mastocytomas and urticaria pigmentosa lesions thus exhibit different patterns of stem cell factor expression. However, a possible pathogenetic role of this factor in mastocytosis remains to be determined.

Phenotypic characterization of skin lesions in urticaria pigmentosa and mastocytomas.

In order to identify possible cellular abnormalities in human mastocytosis, sections from 13 urticaria pigmentosa lesions and 5 mastocytomas were compared with 5 normal skin specimens using histochemical, enzyme histochemical and immunohistochemical techniques. All toluidine blue-positive mast cells also reacted with Fc epsilon RI and c-kit antibodies, almost all stained for tryptase, many for chymase and the myeloid workshop mast cell antibodies, few for Fc epsilon RII and none for the proliferation marker Ki-67. Urticaria pigmentosa lesions contained fewer epidermal Langerhans cells and a lower percentage of avidin-positive mast cells than mastocytomas and normal skin. Mastocytomas exhibited generally weaker staining for mast cell markers and mostly lacked Fc epsilon RI-bound IgE on mast cells and Langerhans cells, although the receptor was able to bind IgE in tissue sections. Most of the mast cell antibodies also reacted with other cell types. Only toluidine blue, avidin, tryptase and chymase stains were mast cell specific. Mast cells in mastocytosis thus differed only to a minor degree from normal mast cells, although distinct pathomechanisms may play a role in urticaria pigmentosa and mastocytosis.

Prognostic significance of mast cells in soft tissue sarcoma.

We counted the number of toluidine blue positive mast cells within and around the tumor in 44 patients with soft tissue sarcomas (STS). Irrespective of their histologic types, these cases were broadly divided into the following two groups: (1) low count (less than 20 mast cells/10 high-power fields [HPF]) and (2) high count (greater than or equal to 20 mast cells/10 HPF). The patients with a high mast cell count showed a significantly better 5-year survival rate than those with a low count (85.9% versus 30.5%; P less than 0.01). Five patients with distant metastases at first admission all belonged to the low count group. These results suggest that the number of mast cells within and around the tumor is a useful prognostic factor for STS.