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PubMed2003

Effects of human mast cell tryptase and eosinophil granule proteins on the kinetics of blood clotting.

Hypereosinophilic syndromes are often associated with thrombosis through unclear mechanisms, and mastocytosis has been associated with a variety of bleeding disorders. The present studies were aimed at defining the roles and interactions of eosinophil and mast cell constituents on the kinetics of blood clotting as measured by thromboelastograms. Eosinophil granule proteins and purified eosinophil peroxidase markedly reduced the anticoagulant properties of the mast cell tryptase/heparin complex. Moreover, eosinophil peroxidase by itself functioned as a powerful procoagulant and also inhibited the anticoagulant actions of heparin in a chromogenic assay for antithrombin III/factor Xa activity. The anticoagulant activity of the tryptase/heparin complex was attributable exclusively to the associated heparin and not to the intrinsic enzymatic activity of tryptase. Eosinophil granule proteins also strongly inhibited the enzymatic activity of tryptase in the presence of hydrogen peroxide, thus implicating a critical role for eosinophil peroxidase. We conclude that eosinophil granule proteins and eosinophil peroxidase both function as powerful procoagulants and also inhibit the anticoagulant and enzymatic activities of mast cell tryptase. The present results thus provide a mechanistic rationale for the well-established link between certain eosinophilic inflammatory disorders and hypercoagulant states. They also suggest that eosinophils may play an important role in neutralizing the anticoagulant activity of mast cell tryptase/heparin in various diseases.

Antithrombin III↗
PubMed2005

Affinity chromatography of tryptases: design, synthesis and characterization of a novel matrix-bound bivalent inhibitor.

beta-Tryptases are mast cell-derived serine proteases that are enzymatically active in the form of an oligomer consisting of four subunits each with trypsin-like activity. The active-site clefts, which are directed toward the central pore of the tetramer, form spatial arrays of four negatively charged S1 binding pockets. Therefore, dibasic inhibitors of appropriate geometry can bind in a bivalent fashion to neighboring subunits. We have recently identified a potent bivalent inhibitor (K(i)=18 nM), based on the bifunctional scaffold cyclo-(-D-Asp-L-Asp-) and the arginine mimetic dl-3-aminomethyl-phenylalanine methyl ester as a ligand for S1 pockets that takes advantage of the this unique tetrameric geometry. To generate an affinity matrix, the bivalent ligand was modified and immobilized on a Sepharose matrix by use of the PEG derivative Jeffamine ED 900 as spacer. This matrix selectively recognizes and binds beta-tryptase from crude protein mixtures and thus is useful as a geometry-driven means of isolating and purifying human mast cell tryptases.

Chromatography, Affinity↗
PubMed1994

The immunohistochemical demonstration of chymase and tryptase in human intestinal mast cells.

An immunohistochemical double-labelling technique for the simultaneous identification of mast cells containing tryptase alone (MCT) or chymase together with tryptase (MCTC) was evaluated quantitatively using two monoclonal antibodies, mAb 1222A (antitryptase) and mAb 1254B (antichymase). Saturation conditions were established for the binding of the antibodies to the mast cell enzymes by counting labelled mast cells in consecutive sections of normal human intestine incubated with serial dilutions of the antibodies. When, under such conditions, the antitryptase was applied after saturation with mAb 1254B, the reproducibility of the double-labelling procedure was excellent. MCT were located preferentially in the intestinal mucosa but, in contrast to what has previously been reported, they were not the predominant type of mast cell at this site. The percentage of MCT of the total number of immunopositive mast cells varied considerably in the colonic mucosa (7-67%, average 30%), while this was not the case in the small intestinal mucosa (5-26%, average 10%). Mast cell chymase, unlike tryptase, was not recognized by the antichymase antibody after aldehyde fixation and a higher apparent fraction of MCT therefore occurred after double labelling. These findings suggest that the proteinase composition of human mast cells, unlike that of murine mast cells, should not be taken as evidence of phenotypic heterogeneity. Taken together with previous observations, they suggest instead that the lack of chymase may be related to functional activity or stage of maturation of the mast cells.

Antibodies, Monoclonal↗
PubMed2005

Application of novel tissue microarrays to investigate expression of tryptase, chymase and KIT protein in placental mast cells.

INTRODUCTION: Tissue microarrays comprise numerous small representative tissue samples from hundreds of different cases assembled on a single histologic slide, and therefore allow high throughput analysis of multiple specimens at the same time. Mast cells are paracrine cells found ubiquitously in connective tissue. Expression of the serine proteases tryptase and chymase, as well as KIT protein, the receptor for stem cell factor (SCF), has been demonstrated in mast cells. Because little is known about the role of mast cells in the placenta, we investigated the number and expression of chymase, tryptase, and KIT protein in placental mast cells using newly developed tissue microarrays. MATERIALS AND METHODS: Tissue microarrays were prepared from archival paraffin tissue blocks of 90 placentae, including 15 normal ones as a control group. Gestational age of the placentae ranged from 7 to 42 weeks. Sections of formalin-fixed paraffin-embedded material were prepared on chemically activated cover-slides. The slides were cut in 4-mm(2) squares containing representative areas, and transferred to a tissue microarray. Hematoxylin and eosin (H&E), chloroacetate esterase (CAE), toluidine blue, periodic acid--Schiff (PAS), and immunohistochemical staining were performed. The number of mast cells and expression of chymase, tryptase, and KIT protein were evaluated in each case. RESULTS: Mast cell numbers in placentae with inflammation/abortion exceeded that of normal placentae. Although statistically not significant, we furthermore observed an increase in chymase-positive mast cells in the group of placentae associated with fetal malformations/chromosomal aberrations compared with normal placentae. DISCUSSION: Novel tissue microarray technique has been introduced into placental research, and allows multiple placental tissue samples to be effectively analyzed simultaneously. This study indicated an increased number of chymase-positive mast cells in placentae with fetal malformation/chromosomal aberration. Activation of angiotensin II by chymase may play a role in fetal malformation. Moreover, it has been speculated that mast cells may only express chymase (MC(C)). Our findings denote the presence of placental MC(C). However, further studies are needed to elucidate more precisely the role of mast cell chymase in the placenta.

Adult↗
PubMed1998

The diagnosis of amniotic fluid embolism: an immunohistochemical study for the quantification of pulmonary mast cell tryptase.

Recent clinical articles have suggested that amniotic fluid embolism (AFE) may be the result of anaphylactic reactions to fetal antigens and that the major part of this clinical syndrome is the result of mast cell degranulation and of the release of histamine, tryptase and other mediators. Tryptase, a neutral protease, is known to be the dominant protein component of the secretory granules of T and TC mast cells. In this paper we have examined the presence and the pulmonary distribution of mast cell tryptase utilizing specific immunohistochemical studies and morphometric evaluation in six cases of fatal amniotic fluid embolism compared to six subjects who died following anaphylactic shock and two control groups (five and six cases respectively) of traumatic death. The results demonstrate a numerical increase of pulmonary mast cells in the subjects who died of AFE (average cell number 54.095) with values corresponding to those encountered in cases of death due to anaphylactic shock (average cell number 51.378) compared with that of the traumatic control groups (average cell number 24.477 and 9.995 respectively). These results can shed light on additional criteria for the diagnosis of amniotic fluid embolism.

Adult↗
PubMed2006

Inhibitory effect of quercetin on tryptase and interleukin-6 release, and histidine decarboxylase mRNA transcription by human mast cell-1 cell line.

Mast cells are involved in inflammatory processes and in allergic reactions where immunologic stimulation leads to degranulation and generation of numerous cytokines and inflammatory mediators. Mast cells have been proposed as an immune gate to the brain, as well as sensors of environmental and emotional stress, and are likely involved in neuropathologic processes such as multiple sclerosis. Among mast cell products, the protease tryptase could be associated with neurodegenerative processes through the activation of specific receptors (PARs) expressed in the brain, while interleukin (IL)-6 likely causes neurodegeneration and exacerbates dysfunction induced by other cytokines; or it could have a protective effect against demyelinisation. In this report we show that quercetin, a natural compound able to act as an inhibitor of mast cell secretion, causes a decrease in the release of tryptase and IL-6 and the down-regulation of histidine decarboxylase (HDC) mRNA from human mast cell (HMC)-1 cells. As quercetin dramatically inhibits mast cell tryptase and IL-6 release and HDC mRNA transcription by HMC-1 cell line, these results nominate quercetin as a therapeutical compound in association with other therapeutical molecules for neurological diseases mediated by mast cell degranulation.

Blotting, Northern↗
PubMed2004

Synthesis of potent and highly selective nonguanidine azetidinone inhibitors of human tryptase.

Azetidinones such as BMS-363131 (2) and BMS-363130 (3), which contain a guanidine group in the C-3 side chain were previously shown to be very potent inhibitors of human tryptase with high selectivity versus other serine proteases, including trypsin. In this letter, we describe the discovery of a number of potent azetidinone tryptase inhibitors in which the guanidine moiety at the ring C-3 position is replaced with primary or secondary amine or aminopyridine functionality. In particular, BMS-354326 (4) is a highly potent tryptase inhibitor (IC(50)=1.8 nM), which has excellent selectivity against trypsin and most other related serine proteases.

Azetidines↗
PubMed2006

Synthesis and evaluation of 4-substituted benzylamine derivatives as beta-tryptase inhibitors.

Since beta-tryptase is considered a critical mediator of asthma, potent tryptase inhibitors may be useful as new agents for the treatment of asthma. We investigated 4-substituted benzylamine derivatives and obtained M58539 (15h) as a potent inhibitor of beta-tryptase (IC50 = 5.0 nM) with high selectivity against other serine proteases, low molecular weight, clog P value less than 5, lack of amidino and guanidino groups, and independence of Zn2+ ion.

Benzylamines↗
PubMed2005

The action of the mast cell product tryptase on cyclooxygenase-2 (COX2) and subsequent fibroblast proliferation involves activation of the extracellular signal-regulated kinase isoforms 1 and 2 (erk1/2).

The mast cell product tryptase, via protease-activated receptor 2 (PAR2), induces cyclooxygenase-2 (COX2) and 15-deoxy-prostaglandin J2 (15d-PGJ2) synthesis. 15d-PGJ2, through the nuclear peroxisome proliferator activated receptor gamma (PPARgamma), subsequently causes fibroblast proliferation. In this study we attempted to determine initial events of the tryptase/PAR2 signaling pathway leading to COX2 induction and fibroblast proliferation. In human fibroblasts (HFFF2), cDNA array, RT-PCR and Western blotting studies demonstrated that tryptase, but not 15d-PGJ2, up-regulates c-jun, c-fos and COX2 expression, and phosphorylates the extracellular signal-regulated kinase isoforms 1 and 2 (erk1/2). Furthermore, tryptase effects on erk1/2, c-jun, c-fos, COX2 and cell proliferation were prevented by PD98059, an inhibitor of the mitogen-activated protein kinase kinase (MEK). Other kinases [P38, stress-activated protein kinase/c-jun N-terminal kinase (SAPK/JUNK), erk5], intracellular Ca(2+) or cAMP were not affected by tryptase/PAR2. Our study identifies crucial intracellular events leading to induction of COX2 and fibroblast proliferation, i.e. a cornerstone of fibrosis.

Base Sequence↗
PubMed2005

The relationship of tryptase- and chymase-positive mast cells to angiogenesis in stage I non-small cell lung cancer.

OBJECTIVE: There are two types of human mast cells, tryptase-positive mast cells (MC(T)) and tryptase- and chymase-positive mast cells (MC(TC)). Although MC(T) have been reported to be related to the generation of angiogenesis, little is known about the involvement of MC(TC) in tumor angiogenesis. In this study, to clarify the relationship between MC(TC) and lung cancer angiogenesis, we evaluated MC(TC), MC(T), and microvessel counts in normal, border, and central lung cancer regions. METHODS: Tumor sections from 32 cases of adenocarcinoma and 13 cases of squamous cell carcinoma were immunostained for chymase to evaluate MC(TC), tryptase to evaluate MC(T,) and CD34 to evaluate microvessel counts. RESULTS: Both MC(TC) and MC(T) counts in the border lung cancer region were significantly higher than in the central region, and the MC(TC) and MC(T) counts in the central region were significantly higher than those in the normal regions. The microvessel counts in the border region were higher than those in the central region. The ratio of MC(TC) to MC(T) in the border region, but not in the central region, was significantly higher than that in the normal region. In the border region, significant correlations not only between MC(T) and microvessel count, but also between MC(TC) and microvessel count were observed. In the central region, a significant correlation between MC(TC) and the microvessel count was observed, but there was no significant correlation between MC(T) and the microvessel count. CONCLUSIONS: These findings suggest that MC(TC) may be involved in the pathogenesis of angiogenesis in lung cancer.

Adenocarcinoma↗
PubMed2004

A possible role of tryptase in angiogenesis in the brain of mdx mouse, a model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterized by muscle degeneration and affects the CNS. Dystrophin is absent in muscle and CNS of both DMD patients and mdx mouse, a model of DMD. While the involvement of vascular compartment in DMD was poorly investigated, some studies suggested a role for mast cells (MC). Tryptase, contained in the MC granules, stimulates angiogenesis in vitro and in vivo. We demonstrated for the first time a correlation between the extent of angiogenesis and the number of tryptase-positive neurons and microvessels and suggest that the tryptase contained in the neurons and in the endothelial cells of the mdx mouse brain may be involved in the regulation of angiogenesis taking place in mdx mouse.

Animals↗
PubMed1999

Histamine and tryptase in nasal lavage fluid after allergen challenge: effect of 1 week of pretreatment with intranasal azelastine or systemic cetirizine.

BACKGROUND: Antihistamines (H1-receptor antagonists) act by competitive antagonism of histamine at H1-receptors. In addition, high concentrations of some antihistamines inhibit allergen-induced histamine release from mast cells in vitro. OBJECTIVE: The purpose of this study was to determine the effect of intranasal azelastine or systemic cetirizine (both potent antihistamines) on the allergen-induced release of mast-cell mediators from the human nasal mucosa in vivo. METHODS: Patients allergic to birch pollen (n = 11) and control subjects not allergic to birch pollen (n = 5) were included in a randomized, double-blind, placebo-controlled, 3-way crossover study outside the pollen season. Each subject was treated with azelastine nasal spray 0.14 mg per nostril twice daily, cetirizine tablets 10 mg every day, or placebo for 1 week using a double-dummy design. At the end of each treatment period, nasal allergen challenges were performed, and the number of sneezes were counted. In addition, nasal lavage fluid was collected, and the levels of mast-cell mediators (histamine and tryptase) were measured. RESULTS: The allergen challenge of patients allergic to pollen produced sneezing and a significant increase in the levels of histamine and tryptase. The challenge of subjects not allergic to pollen produced no such response. Azelastine and cetirizine significantly reduced allergen-induced sneezing and the associated increase in histamine and tryptase levels. No significant differences were found between the azelastine and cetirizine treatments. CONCLUSION: Pretreatment with azelastine or cetirizine inhibits the allergen-induced release of mast-cell mediators from the human nasal mucosa. Our results are consistent with the hypothesis that both antihistamines reduce mediator release from nasal mucosa mast cells in vivo. However, further studies are necessary to test this hypothesis.

Administration, Intranasal↗
PubMed2001

Constitutively raised serum concentrations of mast-cell tryptase and severe anaphylactic reactions to Hymenoptera stings.

Anaphylactic IgE-mediated reactions to Hymenoptera stings vary in their severity for reasons that are not clear. We investigated patients with a history of systemic anaphylatic reactions to honeybee or wasp stings. Nine (75%) of 12 patients with raised tryptase concentrations but only 28 (28%) of 102 patients with lower tryptase concentrations, had a history of severe sting reactions (p=0.004). Raised baseline serum concentrations of mast-cell tryptase and mastocytosis are potential risk factors for severe allergic reactions to Hymenoptera venom.

Adult↗
PubMed2003

Tryptase activates peripheral blood mononuclear cells causing the synthesis and release of TNF-alpha, IL-6 and IL-1 beta: possible relevance to multiple sclerosis.

Presence of mast cells and an increase in the concentration of their products has been reported in multiple sclerosis (MS) plaques. The most abundant secretory mediator of the human mast cell is the tetrameric protease tryptase. We demonstrate that tryptase can activate peripheral mononuclear cells (PBMCs), isolated from healthy donors as well as MS patients for the release of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-1beta. Cytokine secretion was significantly higher in secondary progressive (SP) MS patients and healthy control (HC) individuals than in relapsing-remitting (RR) patients. Our findings suggest that tryptase is, most probably, an important mediator of inflammation in MS.

Adult↗
PubMed2000

The human mast cell tryptase tetramer: a fascinating riddle solved by structure.

Tryptases, the predominant proteins of human mast cells, have been implicated as pathogenetic mediators of allergic and inflammatory conditions, most notably asthma. Until recently, the fascinating properties that distinguish tryptases among the serine proteinases, particularly their activity as a heparin-stabilized tetramer, resistance to most proteinaceous inhibitors, and preference for peptidergic over macromolecular substrates presented a riddle. This review solves this riddle with the help of the crystal structure of the human beta(2)-tryptase tetramer, but also indicates controversies between the unique quaternary architecture and some experimental data.

Amino Acid Sequence↗
PubMed2000

Serum tryptase and the laboratory diagnosis of systemic mastocytosis.

Total tryptase levels of 20 ng/mL or higher in a baseline serum sample when the ratio of total to beta-tryptase is 20 or greater strongly suggest underlying systemic mastocytosis. Whether these criteria prove to be more sensitive than a bone marrow biopsy will require further study. Although the absolute level of total tryptase does not predict disease severity, it may provide a practical method for assessing the efficacy of therapeutic interventions designed to reduce the mast cell burden.

Anaphylaxis↗
PubMed2003

Effective treatment of pruritus in atopic dermatitis using H1 antihistamines (second-generation antihistamines): changes in blood histamine and tryptase levels.

BACKGROUND: Atopic dermatitis (AD) is a common chronic inflammatory and allergic skin disease that almost always begins in childhood and follows a course of remittance and flare-up. AD is characterized by intense pruritus and itchiness that can be triggered by an interplay of genetic, immunologic and environmental factors. Of the mediators, histamine is one of the most potent inducers of pruritus. Serum tryptase, which is also a mediator, may be used to examine allergic disease as well. The development of minimal sedation H1 antihistamines (second-generation antihistamines) has revolutionized treatment of allergic diseases. OBJECTIVE: The present study examines the efficacy of second-generation antihistamines in relieving pruritus due to AD. In addition, the relationship between AD pruritus and antihistamine therapy was analyzed by measuring the blood histamine and tryptase levels. METHODS: Thirty-two AD patients were recruited and underwent second-generation antihistamine therapy for 2 weeks. Seventeen received combined topical corticosteroid treatment (Group 1) and the other 15 did not receive steroid treatment (Group 2). The Severity Index and Pruritus Score were assessed as an AD clinical activity index and compared with baseline data. RESULTS: Both the Severity Index and Pruritus Score improved significantly in Group 1 (P<0.001, P<0.05). Group 2 demonstrated a significant improvement in Pruritus Score (P<0.05), but not in the Severity Index. Plasma histamine levels were significantly higher in AD at baseline compared with healthy controls. CONCLUSION: Following antihistamine therapy, these levels decreased significantly in both AD groups (P<0.05). There was a significant correlation between baseline blood histamine and typtase levels. However, this correlation was not evident following treatment. This may reflect insufficient detection capabilities of the measuring assay. The present results suggest that second-generation antihistamine therapy provides an effective clinical treatment for AD, with a notable improvement in pruritus. Furthermore, antihistamine therapy reduced plasma histamine levels in AD patients. These findings further suggest that high blood histamine and tryptase levels in AD patients contribute to the pathogenesis of this disorder, including the onset of pruritus.

Adult↗
PubMed2003

Neovascularisation, expression of fibroblast growth factor-2, and mast cells with tryptase activity increase simultaneously with pathological progression in human malignant melanoma.

Tissues from 92 proliferative lesions of the melanocytic lineage defining distinct steps in tumour progression were investigated immunohistochemically for changes in angiogenesis, expression of fibroblast growth factor-2 (FGF-2) and density of total mast cells (MCs) and MCs expressing tryptase, an angiogenic-inducing molecule. Although the microvessel number was low in common nevi, it increased significantly in nevi with architectural disorder with varying degrees of melanocytic atypia (termed 'nevi with ADMA'), and these changes persisted during tumour development. Progression of primary melanomas was accompanied by a high microvessel number, and the progression to metastases by another significant increase in the microvessel counts. Expression of FGF-2, evaluated as percentages of positive lesions and positive cells per lesion was upregulated in the course of progression. Changes in expression were associated with nevi with ADMA, tumour changeover, penetration of the tumour into the dermis and metastases. A high correlation was demonstrated in all groups of tissues between the microvessel counts, percentages of FGF-2-positive tumour cells, and both total metachromatic and tryptase-reactive MCs. These results suggest that angiogenesis in human melanoma increases with tumour progression and that FGF-2 secreted by tumour cells and tryptase secreted by host MCs cooperate in its induction.

Adult↗