Mast Cell Granules Research Articles

Extracellular forms of Mycobacterium bovis BCG in the mucosal lymphatic tissues following oral vaccination.

Oral vaccination with BCG provides protective systemic immunity against pathogenic mycobacterial challenge. In this study, the anatomical distribution of Mycobacterium bovis BCG following oral vaccination was investigated. Replicating bacteria in the Peyer's patches and mesenteric lymph nodes were present as solitary rods or clusters of two to three bacteria, the majority of which were isolated ex vivo as extracellular forms. Only a minority were shown to be associated with typical antigen-presenting cells. Acid-fast staining of mast cell granules in lymphoid tissues revealed a potential pitfall for these analyses and may explain previous reports of acid-fast 'coccoid' forms of mycobacteria in tissues.

A Case of Levofloxacin-Induced Anaphylaxis With Elevated Serum Tryptase Levels

Levofloxacin, a fluoroquinolone and L-isomer of the racemate ofloxacin, has been approved for the treatment of acute and chronic bacterial infections. Gastrointestinal complaints are the most frequently reported adverse drug reactions to fluoroquinolones. Other adverse events include headache, dizziness, increased liver enzyme levels, photosensitivity, tachycardia, QT prolongation, and eruptions. Anaphylaxis has been documented as a rare adverse drug reaction to levofloxacin; however, diagnostic tests are needed to evaluate whether these reactions are the result of levofloxacin treatment. While the results of skin tests are considered unreliable due to false-positive responses, the oral provocation test is currently considered to be the most reliable test. Tryptase, a neutral protease, is the dominant protein component of secretory granules in human mast cells, and an increased serum concentration of tryptase is a highly sensitive indicator of anaphylaxis. Herein, we report a case of levofloxacin-induced anaphylaxis in which the patient exhibited elevated serum tryptase levels and a positive oral levofloxacin challenge test result. As anaphylaxis is potentially life-threatening, the administration of fluoroquinolones to patients who have experienced a prior reaction to this type of agent should be avoided.

Tryptase-positive mast cells and angiogenesis in keloids: a new possible post-surgical target for prevention

Literature data indicate that mast cells (MCs) are involved in angiogenesis through the release of several pro-angiogenetic factors among which tryptase, a serine protease stored in MC granules, is one of the most active. However, no data are available concerning the role of MCs during keloids' angiogenesis. In this study, we evaluated the correlations of the number of MCs positive to tryptase (MCDPT) and microvascular density (MVD) within a series of 15 keloids and 10 normotrophic scars, by means of immunohistochemistry and image analysis methods. Data demonstrated a significant difference of MVD and MCDPT between keloids and normotrophic scars and a significant correlation between MVD and MCDPT in keloids. Our results suggest that tryptase-positive MCs might play a key role in keloids' angiogenesis. In this context, several tryptase inhibitors might be clinically evaluated as a possible new anti-angiogenetic approach to prevent keloid formation after surgery.

Mapped! A machinery of degranulation in mast cells. Focus on “Serum- and glucocorticoid-inducible kinase SGK1 regulates reorganization of actin cytoskeleton in mast cells upon degranulation”

<i>Mapped! A machinery of degranulation in mast cells.</i> Focus on “Serum- and glucocorticoid-inducible kinase SGK1 regulates reorganization of actin cytoskeleton in mast cells upon degranulation”

Iron supplementation decreases severity of allergic inflammation in murine lung.

The incidence and severity of allergic asthma have increased over the last century, particularly in the United States and other developed countries. This time frame was characterized by marked environmental changes, including enhanced hygiene, decreased pathogen exposure, increased exposure to inhaled pollutants, and changes in diet. Although iron is well-known to participate in critical biologic processes such as oxygen transport, energy generation, and host defense, iron deficiency remains common in the United States and world-wide. The purpose of these studies was to determine how dietary iron supplementation affected the severity of allergic inflammation in the lungs, using a classic model of IgE-mediated allergy in mice. Results showed that mice fed an iron-supplemented diet had markedly decreased allergen-induced airway hyperreactivity, eosinophil infiltration, and production of pro-inflammatory cytokines, compared with control mice on an unsupplemented diet that generated mild iron deficiency but not anemia. In vitro, iron supplementation decreased mast cell granule content, IgE-triggered degranulation, and production of pro-inflammatory cytokines post-degranulation. Taken together, these studies show that iron supplementation can decrease the severity of allergic inflammation in the lung, potentially via multiple mechanisms that affect mast cell activity. Further studies are indicated to determine the potential of iron supplementation to modulate the clinical severity of allergic diseases in humans.

Assessment of serum tryptase and c-Kit expressing cells from colorectal cancer patients who underwent radical surgery

Background: Data from experimental tumor models suggest that mast cells accumulate near tumor cells before the onset of angiogenesis and that they are required for the macroscopic expansion and metastatic spread of primary tumor cells. Tryptase, a serin protease stored in mast cell granules, plays a role in tumor angiogenesis. Mast cells can release tryptase following c-Kit receptor activation. The aim of this study was to assess serum tryptase levels in colorectal cancer patients before and after radical resection and to quantify c-Kit expressing cells in primary tumor tissue.

Abstract 393: Chymase: Augmented Renal Tubular Protein Expression in Diabetes

Chymase is a serine protease that is predominantly found stored in granules of mast cells. Chymase expression is increased in mesangial cells, podocytes, vascular smooth muscle cells, and cardiomyocytes under hyperglycemic culture conditions. We have presented evidence for enhanced intrarenal chymase-dependent Ang II formation on afferent arteriole vasoconstriction in the diabetic kidney ( Hypertension. 56: e55, 2010). We tested the hypothesis that renal chymase protein expression is enhanced in the diabetic kidney. Kidneys were harvested from littermate control ( db/m ) and type II diabetic ( db/db ) mice at 36-wk-old. Body weight was significantly lower (33 ± 3 vs 40 ± 2 g), while plasma glucose was significantly higher (552 ± 40 vs 220 ± 21 mg/dl) in diabetic (n=6) compared to control (n=7) mice. However, plasma insulin levels were not different from controls (0.77 ± 0.10 vs 0.43 ± 0.11 mg/L), indicating that the diabetic mice are transitioning from type II to type I diabetic disease. Immunohistochemistry was performed using primary rabbit polyclonal antibodies [mMCP-4 (chymase, A Husain), anti-malignant T cell amplified sequence 1 (chymase, Abcam), H + -ATPase (D Brown), and aquaporin 2 (AQP2, J Sands)]. Renal subcapsular and adipose tissue mast cell granules stained positively for toluidine blue and chymase. Chymase protein expression was localized to the apical membrane of connecting tubules and cortical collecting ducts of both control and diabetic kidneys. Chymase was co-localized to AQP2 positive and H + -ATPase negative cells indicating that chymase is specifically expressed in the principal cells of the distal nephron. Prominent chymase immunostaining was observed in the renal papilla and endothelial cells of renal arteries and arterioles in all diabetic, but none of the control kidneys. Chymase was not localized to renal vascular smooth muscle cells. The renal localization of chymase was identical using custom and commercially available polyclonal antibodies. These are the first studies to identify chymase protein expression in the principal cells of the distal nephron. These novel data suggest that the serine protease actions of chymase may be linked to altered epithelial transport mechanisms in the distal nephron in diabetic kidney disease.

Importance of mast cells and their granule serine proteases in microdeformational wound therapy (mdwt) of surgically damaged skin

INTRODUCTION: Stored in the secretory granules of cutaneous mouse mast cells (MCs) are mouse mast cell protease (mMCP)4,mMCP-5, and mMCP-6. Using transgenic mouse lines that lacked these enzymes, it was shown that mMCP-4 and mMCP-5 modulate the outcome of burn-induced injury of the skin. Whether or not these proteases also play prominent roles in the repair of the damaged skin that occurs following surgery remains to be determined.

Polyphosphate Is a Novel Pro-inflammatory Regulator of Mast Cells and Is Located in Acidocalcisomes

Polyphosphate (polyP) is a pro-inflammatory agent and a potent modulator of the human blood-clotting system. The presence of polyP of 60 phosphate units was identified in rat basophilic leukemia (RBL-2H3) mast cells using specific enzymatic assays, urea-polyacrylamide gel electrophoresis of cell extracts, and staining of cells with 4,6-diamidino-2-phenylindole (DAPI), and the polyP-binding domain of Escherichia coli exopolyphosphatase. PolyP co-localizes with serotonin- but not with histamine-containing granules. PolyP levels greatly decreased in mast cells stimulated to degranulate by IgE. Mast cell granules were isolated and found to be acidic and decrease their polyP content upon alkalinization. In agreement with these results, when RBL-2H3 mast cells were loaded with the fluorescent calcium indicator fura-2 acetoxymethyl ester to measure their intracellular Ca(2+) concentration ([Ca(2+)](i)), they were shown to possess a significant amount of Ca(2+) stored in an acidic compartment different from lysosomes. PolyP derived from RBL-2H3 mast cells stimulated bradykinin formation, and it was also detected in human basophils. All of these characteristics of mast cell granules, together with their known elemental composition, and high density, are similar to those of acidocalcisomes. The results suggest that mast cells polyP could be an important mediator of their pro-inflammatory and pro-coagulant activities.

Generation, characterization and structural data of chymase binding proteins based on the human Fyn kinase SH3 domain

The serine protease chymase (EC = 3.4.21.39) is expressed in the secretory granules of mast cells, which are important in allergic reactions. Fynomers, which are binding proteins derived from the Fyn SH3 domain, were generated against human chymase to produce binding partners to facilitate crystallization, structure determination and structure-based drug discovery, and to provide inhibitors of chymase for therapeutic applications. The best Fynomer was found to bind chymase with a KD of 0.9 nM and koff of 6.6x10−4 s−1, and to selectively inhibit chymase activity with an IC50 value of 2 nM. Three different Fynomers were co-crystallized with chymase in 6 different crystal forms overall, with diffraction quality in the range of 2.25 to 1.4 Å resolution, which is suitable for drug design efforts. The X-ray structures show that all Fynomers bind to the active site of chymase. The conserved residues Arg15-Trp16-Thr17 in the RT-loop of the chymase binding Fynomers provide a tight interaction, with Trp16 pointing deep into the S1 pocket of chymase. These results confirm the suitability of Fynomers as research tools to facilitate protein crystallization, as well as for the development of assays to investigate the biological mechanism of targets. Finally, their highly specific inhibitory activity and favorable molecular properties support the use of Fynomers as potential therapeutic agents.

Immunoelectron microscopy study of superficial skin nerves in drug-induced acute urticaria

Few studies have evaluated the ultrastructure of the superficial skin nerves in urticaria. The objective of this study was to describe findings in superficial skin nerves in cases of drug-induced acute urticaria. Seven patients with drug-induced acute urticaria were included in the study. Skin biopsies were obtained from the urticarial lesion and from the apparently normal skin. The 14 fragments collected were processed for immunogold electron microscopy using single stains for antitryptase and anti-FXIIIa antibodies, as well as double immunogold labeling for both. Some sections showed mast cells in the process of degranulation. Following double immunogold staining, 10 nm (FXIIIa) and 15 nm (Tryptase) gold particles were found together throughout the granules in mast cells, indicating that tryptase and FXIIIa are located inside each one of the granules of these cells. Interestingly, we found strong evidence of the presence of tryptase and factor XIIIa in the superficial skin nerves of these patients, both in cases of urticarial lesions (wheals) and in the apparently normal skin. Tryptase and FXIIIa are present in the superficial nerves of the skin in drug-induced acute urticaria. This is the first report of tryptase and FXIIIa expression in the superficial skin nerves of patients with urticaria. Tryptase may be participating in neural activation in these patients, while FXIIIa may be present in the nerves to guarantee the functional integrity of structures.

La degranulation mastocytaire : état des connaissances

Mast cells are immune cells of an hematopoietic origin which mature in peripheral tissues. While they participate in both innate and adoptive immunity as sentinel cells, their major role in medical practice is actually to be found in the effector stage of the allergic response:massive and brutal degranulation leads to the liberation of pre-formed vasoactive, pro-inflammatory and harmful mediators, this event being induced by the interaction of the immunoglobulin E present on RFcɛI membrane receptors with the corresponding multivalent allergen. The preformed mediators of the immediate reaction are stored in the large secretory granules of mast cells. Formation of these granules requires the cooperation of endocytic and secretory pathways, with the biochemical feature of a significant production of proteoglycans which stabilize and protect the granular contents. Degranulatioin induced via IgE and RFcɛI involves translocation of the cytoplasmic granules toward the plasma membrane followed by exocytosis. The general mechanism of vesicular fusion appears to depend on a unique molecular schema: SNARE proteins present on the membranes undergoing fusion form a molecular bridge which keeps the two membranes contiguous; Sec1/Munc18 proteins (SM) control the formation of the SNARE complex and cooperate with it to induce membrane fusion. Entrance of synaptotagmin, a generally vesicular protein which functions as a calcium detector, into the SNARE complex allows the regulation by calcium to begin. The arrival of a complexin stabilizes the SNARE complex while at the same time preventing it from going on to membrane fusion. Here we propose to review current information concerning the mast cell degranulation phenomenon and its regulation.

Primary breast cancer tryptase expression as novel molecular drug target.

e21113 Background: Tryptase, a serine protease stored and released from mast cells granules has been identified as a new non-classical angiogenetic factor. Mast cells can release tryptase following c-Kit receptor activation. We have evaluated the correlations among the number of MCs positive to tryptase (MCDPT), the number of c-Kit receptor expressing cells (C-KREC) and microvascular density (MVD) in a series of 88 primary T1-3, N0-2 M0 female breast cancer by means of immunohistochemistry and image analysis methods. Methods: Six-micrometers thick serial sections of formalin-fixed and paraffin-embedded bioptic tumor samples were microwaved at 500 W for 10 min. and treated with a 3% hydrogen peroxide solution. Sections were incubated with primary antibodies: anti-tryptase (AA1; Dako, Glostrup, Denmark), anti-c-Kit receptor (A4502;Dako, Glostrup, Denmark) and anti-CD34 (QB-END 10; Bio-Optica Milan, Italy). In serial sections MVD, MCDPT and C-KREC were counted by means of image analysis at x400. Results: Data demonstrated a significantly (r= ranging from 0.71 to 0.91; p: ranging from 0.001 to 0.003 by Pearson’s analysis respectively) correlation between MVD, MCDPT and C-KREC to each other. Conclusions: Published in vitro data suggest that tryptase induce angiogenesis in vascular endothelial cells and breast cancer cells lines. According to these data we shown that MVD, MCDPT and C-KREC paralleled to each other suggesting a role in in vivo breast cancer angiogenesis. In this context several tryptase inhibitors such as gabexate mesilate or nafamostat mesilate might be evaluated in clinical trials as a new antiangiogenetic approach.

Is serum tryptase level a novel biomarker in colorectal cancer patients?

e21134 Background: Tryptase is a serin protease stored in mast cell granules that plays a role in tumour angiogenesis. Mast cells (MCs) can release tryptase following c-Kit receptor activation. On the other hand colo-rectal cancer (CRC) is a well-established angiogenesis dependent tumour and anti-angiogenic based therapy is a standard treatment in metastatic CRC. This preliminary study aims to assess tryptase serum levels in 51 CRC patients before and after radical surgery. Methods: In this study patients with stage B and C CRC (according to Astler e Coller staging system) were selected. Samples of blood were taken from CRC patients between 7 and 9 a.m.1 day before and 1 day after curative surgery. Venous blood was dispensed into a tube for serum (Becton Dickinson Hemogard Vacutainer Systems, Plymouth, UK). Tryptase levels were measured using the UniCAP Tryptase Fluoroenzymeimmunoassay (Pharmacia,Uppsala, Sweden). Primary tumour tissue were also evaluated in terms of MCs positive to tryptase by immunohistochemistry utilizing an anti-tryptase primary antibody (clone AA1; Dako, Glostrup, Denmark). Results: Mean ± s.d. tryptase level pre-tumour surgical resection was 6.37 ± 4.57 μg/L, and mean ± s.d. tryptase level post tumour surgical resection was 5.15 ± 3.82 μg/L. A statistically significant difference between pre-tumour surgical resection and post-tumour surgical resection tryptase level concentrations was found: p=0.000 by t-test. A statistically significant correlation between MCs positive to trypase and serum tryptase levels pre-surgical resection was also found (r=086; p=0.000) by Pearson’s analysis. Conclusions: This pilot report analyzes the possible significance of serum tryptase levels changes in CRC patients who underwent curative surgery. Our results demonstrated higher serum tryptase levels CRC patients suggesting the release of tryptase from MCs of primary CRC tissue. As expected, after radical surgical resection, serum tryptase levels had decreased. We suggest that tryptase may play a role as a new predictive biomarker in CRC patients. In this context several tryptase inhibitors such as gabexate mesilate and nafamostat mesilate might be evaluated as adjuvant treatment in clinical trials.

Dermal dendrocytes FXIIIA+ phagocytizing extruded mast cell granules in drug‐induced acute urticaria

Few authors have been attempting between mast cells and dermal dendrocytes interactions on urticaria. To describe the extruded mast cell granules and dermal dendrocytes in drug-induced acute urticaria. Seven patients with drug-induced acute urticaria were enrolled in the study. We token skin biopsies of urticaria lesion and perilesional skin. The 14 fragments collected were processed to immunogold electron microscopy using single stains to tryptase and FXIIIa, besides double immunogold labeling with both. Some sections demonstrated mast cells in degranulation process, both in anaphylactic and piecemeal degranulation types. After double immunogold staining, 10 nm (FXIIIa) and 15 nm (Tryptase) gold particles were present together over the granules in mast cells indicating that tryptase and FXIIIa are each localized within the granules of these cells. Interestingly, we found a strong evidence of than the exocytosed mast cell granules contents both FXIIIa and tryptase immunolabeled are phagocytized by dermal dendrocytes. The current observations provide morphological evidence that the exocytosis-phagocytosis mechanisms of mast cell granules represents one pathophysiological example of mast cells-dermal dendrocytes interactions in urticaria.

Localization of heat shock protein 70 in rat mast cells

Heat shock proteins (Hsp) are intracellular chaperons, as well as extracellular molecules with immunomodulatory and signaling functions engaged in adaptation to stress on the cellular and organism levels. The presence of Hsp in secretory granules of mast cells (MCs) may be correlated with mast cells’ active participation in adaptation to stress. Using immunoelectron microscopy, we showed that Hsp70 was localized in secretory granules of rat pericardial and peritoneal mast cells. Localization of Hsp70 in rat peritoneal mast cells isolated by Percoll density gradient centrifugation was confirmed by immunoblotting. The possible involvement of mast cells in production of extracellular Hsp70, as well as Hsp70 functions inside the mast cells, is discussed.

Impact of Mast Cell Chymase on Renal Disease Progression

Chymase, a serine protease found in mast cell granules, is released into the interstitium following injury or inflammation. Chymase is the primary ACE-independent pathway of angiotensin II formation, and also functions to activate TGF-beta and other promoters of extracellular matrix degradation, thereby playing a role in tissue remodeling. In the diseased kidney, chymase-containing mast cells markedly increase and their density correlates with tubulointerstitial fibrosis severity. Studies in humans support the pathologic role of chymase in diabetic nephropathy, while animal studies form the basis for the importance of increased chymase-dependent angiotensin II formation in progressive hypertensive, diabetic and inflammatory nephropathies. Moreover, humans with kidney disease express chymase in diseased blood vessels in concordance with significantly elevated plasma chymase levels. Conversely, specific chymase inhibitors attenuate angiotensin II production and renal fibrosis in animal models, suggesting their potential therapeutic benefit in human nephropathy, where chymase-containing mast cells accumulate and contribute to progressive disease.

Nanoscale Mimics of Mast Cell Messengers

Synthetic analogs of mast cell granules have been engineered to deliver cytokines to lymph nodes and improve the performance of vaccines.

Mast Cell Restricted Mouse and Human Tryptase·Heparin Complexes Hinder Thrombin-induced Coagulation of Plasma and the Generation of Fibrin by Proteolytically Destroying Fibrinogen

The mouse and human TPSB2 and TPSAB1 genes encode tetramer-forming tryptases stored in the secretory granules of mast cells (MCs) ionically bound to heparin-containing serglycin proteoglycans. In mice these genes encode mouse MC protease-6 (mMCP-6) and mMCP-7. The corresponding human genes encode a family of serine proteases that collectively are called hTryptase-β. We previously showed that the α chain of fibrinogen is a preferred substrate of mMCP-7. We now show that this plasma protein also is highly susceptible to degradation by hTryptase-β· and mMCP-6·heparin complexes and that Lys(575) is a preferred cleavage site in the protein α chain. Because cutaneous mouse MCs store substantial amounts of mMCP-6·heparin complexes in their secretory granules, the passive cutaneous anaphylaxis reaction was induced in the skin of mMCP-6(+)/mMCP-7(-) and mMCP-6(-)/mMCP-7(-) C57BL/6 mice. In support of the in vitro data, fibrin deposits were markedly increased in the skin of the double-deficient mice 6 h after IgE-sensitized animals were given the relevant antigen. Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Our discovery that mouse and human tetramer-forming tryptases destroy fibrinogen before this circulating protein can be converted to fibrin changes the paradigm of how MCs hinder fibrin deposition and blood coagulation internally. Because of the adverse consequences of fibrin deposits in tissues, our data explain why mice and humans lack a circulating protease inhibitor that rapidly inactivates MC tryptases and why mammals have two genes that encode tetramer-forming serine proteases that preferentially degrade fibrinogen.

Tailor-made mast-cell granules

Mast cells induce protective immune responses through secretion of stimulatory granules. Microparticles modelled after mast-cell granules are now shown to replicate and enhance the functions of their natural counterparts and to direct the character of the resulting immunity.