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PubMed1997

[Pterygium and mast cells--mast cell number, phenotype, and localization of stem cell factor].

We examined the number and phenotype of mast cells, and the localization of stem cell factor (SCF) as a growth factor of mast cells in the excised tissue of 38 cases of pterygium. In histopathology with toluidine blue stain and immunohistochemistry with a monoclonal antibody to tryptase, the mean mast cell count in pterygium specimens was twice as high as in normal conjunctiva. In pterygium specimens more than 94% of tryptase-positive mast cells were found to express chymase and c-kit. There was no phenotypic difference between mast cells in pterygium and normal conjunctiva. In all immunohistochemical specimens in which we could examine the head of the pterygium, SCF was expressed in subepithelial fibroblasts at the central edge of pterygium. The results suggest that overexpression of SCF was accompanied with the augmentation of mast cells in the pterygium.

Cell Count↗
PubMed1997

Systemic mast cell disease (mastocytosis). General aspects and histopathological diagnosis.

Systemic mast cell disease/mastocytosis (SMCD) is best defined as a multitopic proliferation of cytologically and/or functionally abnormal tissue mast cells (TMC). SMCD preferentially involves the bone marrow, skin, spleen, liver, and lymph nodes. The histopathological diagnosis of SMCD may be very difficult to make, and the disease is often not considered in the differential diagnosis of lymphoreticular neoplasia. In suspected cases of SMCD, basic dyes such as Giemsa and toluidine blue are useful to demonstrate the specific metachromatic granules of TMC. The naphthol AS-D chloroacetate esterase reaction has also proved to be very reliable for enzyme-histochemical identification of TMC. Major diagnostic problems may arise in cases of malignant or "aggressive" SMCD exhibiting tissue infiltrates consisting predominantly of highly atypical, non-metachromatic TMC, which are usually also only weakly reactive for chloroacetate esterase. Immunostaining with antibodies against the mast cell-specific proteases tryptase and chymase has proved to be of great value of establishing the correct diagnosis in such cases. Anti-tryptase antibodies have major diagnostic significance due to their extremely high sensitivity and specificity. The classification of SMCD is controversial, but there is increasing support for the differentiation of at least two major subtypes that differ in prognosis: (i) a benign or "indolent" variant in which skin involvement (urticaria pigmentosa-like skin lesions) is usual, but associated malignant hematological disorders are rare; and (ii) a malignant or "aggressive" variant where skin involvement is usually absent but concomitant malignant hematological disorders (myelodysplastic and myeloproliferative syndromes and acute non-lymphocytic leukemias) are very common. Preliminary molecular biological studies of a few cases of malignant SMCD using the recently developed HUMARA assay have yielded evidence that the disease is monoclonal.

Animals↗
PubMed1998

Diagnosis and pathogenesis of the anaphylactic and anaphylactoid reactions to anaesthetics.

Immediate adverse reactions to anaesthetics have an immune mechanism in more than 50% of the cases. They are mainly due to muscle relaxant drugs. A prospective evaluation of tryptase, histamine and serotonin for diagnosing anaphylaxis to anaesthetics was performed over 2 years. The sensitivity of each marker was at 60-70% and it reached 80% when combining tryptase and histamine. Specific IgE have been already observed in serum from patients allergic to muscle relaxant, thiopentone, morphine, phenoperidine, propofol and radio-contrast media. However, the recent progress in the identification of drug epitopes by Sepharose-solid drug phase IgE radioimmunoassay has to be reconsidered as non-specific binding of hydrophobic drugs such as propofol to hydrophobic serum IgE has been observed recently in patients with drug allergy. In addition, association of drugs such as propofol and muscle relaxant may potentiate the mediator release by a non-elucidated mechanism.

Anaphylaxis↗
PubMed1998

Human mast cells express stem cell factor.

Stem cell factor (SCF) is a major cytokine regulator of mast cell growth and function. The present study demonstrates that human mast cells are able to produce SCF. Constitutive synthesis of SCF mRNA was seen in the mast cells isolated from human lung and skin by RT-PCR. This was confirmed by in situ hybridization in conjunctival mast cells of both tryptase-only (MCT) and tryptase/chymase (MCTC) subsets. SCF protein product was found in conjunctival MCT and MCTC mast cells by immunohistochemistry. Soluble SCF protein was detected in the culture supernatant of isolated lung mast cells by ELISA, and cross-linkage of IgE receptor (Fc epsilon-RI) on the lung mast cells in culture did not alter SCF mRNA expression, or the secreted soluble SCF protein. This was consistent with the finding that levels of SCF mRNA expression in conjunctival mast cells were similar between normal subjects and patients with seasonal allergic conjunctivitis (SAC). This study shows that human mast cells themselves are a cellular source of SCF, as well as being target cells for this growth factor. SCF may regulate mast cell growth and function via both paracrine and autocrine mechanisms. The production of SCF by mast cells may be regulated via mechanisms other than IgE receptor-mediated pathways.

Cell Culture Techniques↗
PubMed2000

Mast cell involvement in normal human skin wound healing: expression of monocyte chemoattractant protein-1 is correlated with recruitment of mast cells which synthesize interleukin-4 in vivo.

Mast cells (MCs) are known as key cells of immediate type hypersensitivity reactions. It has recently been shown that MCs regulate fibroblast proliferation by heterotypic cell-cell contact and secretion of interleukin-4 (IL-4) in vitro. It was therefore hypothesized that MCs may contribute to wound repair in vivo. Using immunohistology and in situ hybridization, the time course of mast cell recruitment and the expression of MC-attractant chemokines were analysed in a human skin wound-healing model, and the production of IL-4 by MCs in vivo was investigated. The data obtained indicate that the five-fold increase of the tryptase+ MCs at the fibrotic border of the wound within the first 10 days is the result of increased recruitment/survival of MCs or MC precursors, but not of increased local proliferation. Recruitment of MCs is paralleled by the expression of monocyte chemoattractant protein-1 (MCP-1), but not by other chemokines such as RANTES (regulated on activation, normal T cell expressed and secreted) and/or MIP (macrophage inflammatory protein)-1alpha/beta. Notably, 60-70% of MCs exhibited strong and selective IL-4 immunoreactivity, whereas other resident and passenger cells were rather quiescent. The data suggest that MC contribute significantly to the cytokine network of wound repair via MC-derived IL-4 and stimulation of fibroblast proliferation.

Adult↗
PubMed2000

Murine mast cell lines as indicators of early events in mast cell and basophil development.

To study early events in mast cell / basophil development, the phenotype of a panel of murine cell lines at various stages of differentiation was determined. Based on the expression on various mast cell-specific proteases and several additional hematopoietic differentiation markers, the cell lines CFTL-15 and MCP5 / L were clearly identified as mast cells, although with a relatively immature phenotype. These two cell lines express the high-affinity IgE receptor alpha-chain, the mouse mast cell protease (MMCP)-5 and the carboxypeptidase A (CPA). Bone marrow-derived mast cells and the transplantable mast cell tumor MTC were shown to express the IgE receptor alpha-chain, MMCP-5 and CPA, as well as the mast cell tryptase MMCP-6 and the chymase MMCP-4, a protease expressed only during late stages of mast cell differentiation. These two cell types thus display a more mature mast cell phenotype. In contrast, the cell lines P815 and 32D cl3 did not express any mast cell differentiation markers. Interestingly, the IC-2 cell line was shown to express several markers for immature mast cells and in addition MMCP-8, a serine protease which may represent a marker for mouse basophils. By antibody staining, almost all IC-2 cells were shown to express MMCP-8. This indicates that individual cells may simultaneously express both mast cell and basophil markers. Moreover, these findings suggest that an early branch point in hematopoietic development where mast cells and basophils have a common precursor cell may exist.

Animals↗
PubMed2005

Clinical significance of vascular endothelial growth factor-C and vascular endothelial growth factor receptor 3 in patients with T1 lung adenocarcinoma.

BACKGROUND: Vascular endothelial growth factor-C (VEGF-C) plays an important role in lymphangiogenesis and activates VEGF receptor-3 (VEGFR-3). Lymphatic spread is an important prognostic factor in patients with lung adenocarcinoma. The aim of the current study was to determine whether the expression of VEGF-C and VEGFR-3 correlates with clinicopathologic factors and prognosis in patients with TNM classification T1 lung adenocarcinoma. METHODS: The authors conducted a retrospective review of 129 consecutive patients who underwent complete resection for T1 lung adenocarcinoma. Immunohistochemical staining for VEGF-C, VEGF, VEGFR-3, CD34 (microvessels), tryptase (mast cells), and CD68 (macrophages) was performed to statistically analyze clinicopathologic implications of VEGF-C and VEGFR-3 status. RESULTS: Of 129 patients with T1 lung adenocarcinoma, 56 (43.3%) patients were positive for tumor-cell VEGF-C and 73 (56.6%) and 69 (53.5%) patients were positive for tumor-cell and endothelial-cell VEGFR-3, respectively. Patients with positive staining for tumor-cell VEGF-C showed significantly less favorable survival rates than patients with negative staining (P = 0.031). The survival rates of patients with positive staining for tumor-cell and endothelial-cell VEGFR-3 were significantly lower than those with negative staining (P = 0.0034 and P = 0.0020, respectively). Patients with positive staining for both tumor-cell VEGF-C and endothelial-cell VEGFR-3 exhibited the most unfavorable prognoses. Multivariate analysis demonstrated that coexpression of tumor-cell VEGF-C and endothelial-cell VEGFR-3 was an independent negative prognostic factor (P = 0.0129) as well as N factor (P = 0.0020). CONCLUSIONS: VEGF-C and VEGFR-3 status may be indicative of survival rates for patients with T1 lung adenocarcinoma.

Adenocarcinoma↗
PubMed1995

Expression of the c-kit gene product in normal and neoplastic mast cells but not in neoplastic basophil/mast cell precursors from chronic myelogenous leukaemia.

The expression of the c-kit gene product has been examined in normal mast cells, mast cell neoplasms, and basophil/mast cell precursors obtained from patients with chronic myelogenous leukaemia (CML). Formalin-fixed, paraffin-embedded sections or smears fixed with formalin vapour were studied by immunohistochemical methods, using a polyclonal antibody against the c-kit gene product. Normal and neoplastic mast cells showed a positive immunoreaction for c-kit gene product, but neoplastic basophil/mast cell precursors from CML patients lacked c-kit gene product by immunohistochemical and flow cytometric methods, even in cells having mast cell granules, together with or without basophil granules. Mast cell tryptase was, however, expressed in normal and neoplastic mast cells and basophil/mast cell precursors containing mast cell granules. In addition, cells of monocyte/macrophage lineage lacked c-kit gene product. These findings indicate that the c-kit gene product may play an important role in the development and function of mast cell but not of cell of basophil and monocyte/macrophage lineage.

Animals↗
PubMed2000

High-throughput screening of enzyme inhibitors: automatic determination of tight-binding inhibition constants.

Determination of tight-binding inhibition constants by nonlinear least-squares regression requires sufficiently good initial estimates of the best-fit values. Normally an initial estimate of the inhibition constant must be provided by the investigator. This paper describes an automatic procedure for the estimation of tight-binding inhibition constants directly from dose-response data. Because the procedure does not require human intervention, it was incorporated into an algorithm for high-throughput screening of enzyme inhibitors. A suitable computer program is available electronically (http://www.biokin.com). Representative experimental data are shown for the inhibition of human mast-cell tryptase.

Algorithms↗
PubMed1993

Catabolism of intact type VI collagen microfibrils: susceptibility to degradation by serine proteinases.

We present the first direct biochemical evidence for the turnover of intact type VI collagen microfibrils. Matrix-degrading enzymes of the serine proteinase class, including rat mast cell chymases I and II, human mast cell tryptase, neutrophil elastase, cathepsin G and trypsin, were able to catabolize intact type VI collagen microfibrils isolated from foetal bovine skin and metabolically labelled intact type VI collagen immunoprecipitated from fibroblast culture medium. By contrast, intact type VI collagen was not degraded by the human matrix metalloproteinases, MMP-1, MMP-2, MMP-3 and MMP-9. These data have important implications for the stability of type VI collagen in connective tissues and highlight the potential role of serine proteinases both in normal type VI collagen turnover and in inflammatory conditions characterized by matrix degradation.

Animals↗
PubMed1996

IgE-anti-IgE-induced prostaglandin D2 release from cultured human mast cells.

To elucidate the characterization of cultured human mast cells, the generation of prostaglandin D2 (PGD2) upon stimulation with IgE-anti IgE challenge was determined by using gas chromatography/mass spectrometry/selected ion monitoring (GC/MS/SIM) assay. Mononuclear cells obtained from human umbilical cord blood were cultured in the presence of Steel Factor, IL-6, and PGE2. After 8 weeks of culture, approximately 90 to 95% of cultured cells became tryptase-positive and included basophilic granules. At 12 weeks of culture, cells were harvested and incubated with 1 microgram/ml of human IgE for 1 hr and then challenged with 10 micrograms/ml of anti-IgE. The level of PGD2 release into the supernatant was measured by GC/MS/SIM with the stable isotope dilution method. Cyclooxygenase inhibitors affected the PGD2 release from the cell by IgE-anti IgE challenge incubation. Indomethacin inhibited 98% of PGD2 release. Acetylsalicylic acid (aspirin) and NS-398 (potent COX-2 inhibitor) also inhibited PGD2 release by 85 and 45% respectively.

Antibodies, Anti-Idiotypic↗
PubMed2000

Extrapancreatic trypsin-2 cleaves proteinase-activated receptor-2.

Proteinase-activated receptors (PARs) are activated by proteolytic removal of a short amino terminal peptide, thus exposing a new amino terminus that functions as a tethered ligand that activates the receptor. With the aim to identify and study potential activators of PAR-2 we have developed a new method to measure proteolytic cleavage of PARs. PAR-2 was tagged with the insulin C-peptide that upon receptor cleavage is released and quantified using an ELISA. The modified receptor, shown to be functional in mouse 3T3 cells, was expressed in an insect cell line and the ability of different proteinases to cleave PAR-2 was studied. Two different mast cell tryptases cleaved PAR-2 in a concentration dependent manner, but were much less potent than pancreatic trypsin and trypsin-2 isolated from a carcinoma cell line. Pancreatic trypsin and trypsin-2 were almost equally effective at cleaving PAR-2 suggesting that extrapancreatic trypsins are potential in vivo activators of PAR-2.

3T3 Cells↗
PubMed2001

Protease-activated receptor-2 (PAR2) in the airways.

Protease-activated receptors (PARs) act as sensors for active extracellular serine proteases. Since serine proteases like mast cell tryptase are associated with inflammatory processes, PARs may represent novel pharmacological targets in airway diseases like asthma and chronic obstructive pulmonary disease. However, our present understanding of the physiological roles of PARs is in its infancy. In this review we highlight evidence for the involvement of PARs in airway disease and propose that these novel receptors may play mainly protective roles.

Asthma↗
PubMed1996

Inhibitory effect of pulmonary surfactant on Sendai virus infection in rat lungs.

Intranasal infection of rats with active (infectious) Sendai virus enhances secretion of tryptase Clara, a Sendai virus-activating protease, into the bronchial lumen by Clara cells of the bronchial epitheliums, and inversely suppresses secretion of pulmonary surfactant, an inhibitor of the protease, into the lumen [Kido H et al. (1993) FEBS Lett 322: 115-119]. A trypsin-resistant mutant, TR-2, showed similar effects, although its replication was restricted to a single cycle in the lungs. In contrast, neither nonactive (noninfectious) wild-type virus possessing receptor-binding activity and lacking envelope fusion activity nor UV-inactivated virus retaining receptor binding and envelope fusion activities altered the mode of secretions. These results indicate that viral replication is required for producing a condition in the bronchial lumen for proteolytic activation of progeny virus, thereby infection is extended to a fatal pneumonia. On the other hand, intranasal administration of infected rats with pulmonary surfactant suppressed activation of progeny virus and pathological changes in the lungs, suggesting a therapeutic use of pulmonary surfactant for influenza pneumonia.

Animals↗
PubMed2005

Mast cell hyperplasia induced by multiple challenge with cedar pollen in sensitised guinea pigs.

OBJECTIVE AND DESIGN: Because immediate allergic symptoms are aggravated by repetitive antigen challenges in a guinea pig model of Japanese cedar pollen-induced conjunctivitis, we determined whether conjunctival mast cells are different in number between the acute and chronic stages. METHODS: Sensitised guinea pigs were challenged by dropping a pollen suspension into their eyes once a week. Conjunctival tissue sections were stained with toluidine blue. Ophthalmic lavage was performed to assay for mast cell mediators. RESULTS: At the 20th and 40th challenges, the number of mast cells increased by 4- to 5-fold compared with the 1st challenge. Although mast cell degranulation was insignificant 10 min after the 1st challenge, the 20th and 40th challenges produced significant degranulation. After multiple challenges, the amount of histamine and tryptase-like activity in the lavage fluid was dramatically increased. CONCLUSION: Increased mast cells are associated with aggravated symptoms. Mast cell mediators may be involved in pathogenesis at the chronic stage.

Acute Disease↗
PubMed2005

[Anaphylactoid reaction in occult systemic mastocytosis. A rare dermatologic emergency ].

With the occurrence of unclear consciousness conditions primarily internal or neurological causes are considered. Of a systemic mastocytosis one thinks only rarely, which can accompany without or with slight clinically visible skin changes. In the following we report on a patient who has repeated unclear shock conditions and required resuscitation several times without a recognizable cause, with whom a systemic mastocytosis could be proven. Clinically very discrete lesions of mastocytosis were recognizable from the skin. Only an increased tryptase level referred to being present an occult systemic mastocytosis. The diagnostics, potential triggers and therapy of this disease are to be discussed on the basis the available case.

Adult↗
PubMed2001

Characterization of the gene encoding mouse mast cell protease 8 (mMCP-8), and a comparative analysis of hematopoietic serine protease genes.

Serine proteases are important granule constituents in several of the major hematopoietic cell lineages. We present here the nucleotide sequence of the gene encoding mouse mast cell protease 8 (mMCP-8). mMCP-8 was initially isolated as a cDNA from a mouse mast cell line, but has recently been found to be expressed primarily by mouse basophils. mMCP-8 and its rat homologues, rMCP-8, -9, and -10, form a new group of mast cell/basophil proteases, which are more closely related to the T-cell granzymes and neutrophil cathepsin G than to the mast cell tryptases and chymases. A dot matrix comparison of the mMCP-8 gene with other closely related hematopoietic serine protease genes shows detectable homology only in the exonic regions of the genes. No indication for conservation in the promoter region or introns was observed. This latter finding indicates that the upstream regulatory region has evolved at a relatively high rate. However, despite the low degree of direct sequence conservation, no major differences in the sizes of introns or exons were observed between mMCP-8 and genes for the closest related hematopoietic serine proteases, the mouse T-cell granzymes and cathepsin G, indicating that after evolutionary separation from the T-cell granzymes and cathepsin G, the majority of mutations primarily involved single base pair substitutions or short insertions or deletions.

Amino Acid Sequence↗
PubMed2004

Regulation of mast cell characteristics by cytokines: divergent effects of interleukin-4 on immature mast cell lines versus mature human skin mast cells.

Mast cells (MC) are of hematopoietic origin but complete their differentiation exclusively within tissues. The mediators that positively or negatively affect the maturation process are incompletely defined. Here, the human MC line HMC-1 (subclone 5C6) was used along with several treatments (IL-4, IL-6, NGFbeta), either alone or in combination, and MC differentiation was monitored by flow-cytometric analysis of c-kit, tryptase, and FcepsilonRIalpha expression. Of the different treatments, IL-4 displayed the clearest effects by suppressing the expression of the three markers and inhibiting cellular growth, while the other cytokines had no (NGFbeta) or negligible (IL-6) effects only. The downregulating effects of IL-4 could not be overcome by any other treatment. There is some controversy in the literature as to the impact of IL-4 on the MC lineage. To determine whether the effects from IL-4 were differentiation stage dependent, two further human MC subsets (skin MC and LAD 2 cells) were investigated. No effects on c-kit and FcepsilonRIalpha expression were noted when terminally differentiated skin MC were used as target cells, while a modest downregulation of c-kit was observed with intermediately matured LAD 2 cells. In sharp contrast to HMC-1 5C6 cells, the survival of skin MC was significantly enhanced by IL-4 treatment. Our data therefore imply that at a lower maturation stage, IL-4 acts as a negative regulator of the MC lineage, but that this property disappears or is even reversed upon terminal differentiation of the cell. Our study provides direct proof that the effects of IL-4 vary substantially in the course of MC maturation.

Antigens, CD↗