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PubMed1996

Characteristics of histamine release from cultured human mast cells.

BACKGROUND: The mast cell is one of the important cells in the pathogenesis of allergic disorders. However, isolating human mast cells is a laborious procedure. Recently, cultured human mast cells raised from umbilical cord blood cells have become available. It is necessary to examine whether these cells are useful in investigating the role of mast cells in human diseases. OBJECTIVE: The phenotype of mast cells depends on their anatomical sites. To examine which phenotype of mast cells these cultured mast cells most closely resemble, their ability to release was investigated. METHODS: The mast cells were raised from human umbilical cord blood cells in the presence of stem cell factor and interleukin-6. To determine the mast cell subtypes, the mast cells were immunocytochemically stained for tryptase and chymase. The cultured mast cells were then stimulated with various secretagogues, and histamine release was measured by a fluorometric technique using high-performance liquid chromatography. RESULTS: The immunocytochemical staining for mast cell proteases revealed that virtually all cells contained tryptase, the definitive marker of mast cells, and that about a quarter of the cells contained chymase. Anti-IgE effectively stimulated these mast cells to release histamine in a dose-dependent, time-dependent manner. The release was completed in about 30 min. One of the non-specific stimuli, calcium ionophore A23187, also induced histamine release in a dose-dependent, time-dependent manner. In contrast, compound 48/80 and substance P failed to induce histamine release from these cells. CONCLUSION: Cultured human mast cells resemble lung mast cells in their ability to release histamine. They will help in studying the functional properties of human mast cells and may contribute to clarifying the pathophysiology of human allergic diseases.

Antibodies, Anti-Idiotypic↗
PubMed1996

Eosinophil granule proteins activate human heart mast cells.

Eosinophilia in humans is often associated with heart disease and cardiac localization of eosinophil granule proteins, and several results suggest that granule proteins mediate endomyocardial damage. Here we investigated the in vitro effects of the four principal eosinophil granule proteins (eosinophil cationic protein (ECP), major basic protein (MBP), eosinophil-derived neurotoxin, and eosinophil peroxidase (EPO)) on the activation of effector cells of inflammation (mast cells) isolated from human heart tissue (HHMC). ECP and, to a lesser extent, MBP (0.3-3 microM), but not eosinophil-derived neurotoxin and eosinophil peroxidase stimulated the release of preformed (histamine and tryptase) and the de novo synthesis of vasoactive and proinflammatory mediators (PGD2) from HHMC. Activation of HHMC by ECP and MBP was Ca2+- and temperature-dependent and was abolished by preincubation (15 min, 37 degrees C) with 2-deoxy-D-glucose (10 mM) and antimycin A (1 microM). There was a significant correlation between the maximal percentage of histamine release induced by ECP and anti-IgE from HHMC (rs = 0.73; p < 0.005), by MBP and anti-IgE (rs = 0.79; p < 0.001), and by ECP and MBP (rs = 0.65; p < 0.005). A positive correlation was also found between histamine and tryptase secretion (rs = 0.71; p < 0.001) and between histamine and PGD2 release induced by ECP from HHMC (rs = 0.85; p < 0.001). This is the first demonstration that some eosinophil cationic proteins, namely ECP and MBP, found at the site of heart damage in patients with eosinophilia, act as complete secretagogues on HHMC. This observation indicates another mechanism by which infiltrating eosinophils and their metabolic products cause inflammatory reactions and thus endomyocardial lesions in patients with eosinophilia.

Blood Proteins↗
PubMed1996

Characterization of the ocular antiallergic and antihistaminic effects of olopatadine (AL-4943A), a novel drug for treating ocular allergic diseases.

Olopatadine (AL-4943A; KW-4679) [(Z)-11-[3-(dimethylamino)propylidene]-6, 11-dihydrodibenz[b,e]oxepine-2-acetic acid hydrochloride] is an antiallergic/antihistaminic drug under development for topical ocular use. The effects of the compound on release of proinflammatory mediators (histamine, tryptase and prostaglandin D2) from monodispersed human conjunctival mast cells were assessed. Histamine receptor subtype binding affinities and functional potencies were determined with ligand binding and phosphoinositide turnover assays, respectively. Olopatadine inhibited the release of histamine, tryptase and prostaglandin D2, in a concentration-dependent manner (IC50 = 559 microM). Evaluation of the interaction of olopatadine with histamine receptors revealed a relatively high affinity for the H1 receptor (Ki = 31.6 nM, pKi = 7.5 +/- 0.1, n = 7) but lower affinities for H2 receptors (Ki = 100 microM, pKi = 4.0 +/- 0.19, n = 7) and H3 receptors (Ki = 79.4 microM, pKi = 4.1 +/- 0.16, n = 7). The H1 selectivity of olopatadine was superior to that of other ocularly used antihistamines studied, such as ketotifen, levocabastine, antazoline and pheniramine. The profiling of olopatadine in 42 nonhistamine receptor binding assays revealed that olopatadine interacts with only two nonhistamine receptor/uptake sites to any significant degree (pIC50 < or = 5-6). Olopatadine inhibited histamine-induced phosphoinositide turnover in human conjunctival epithelial cells (IC50 = 10 nM, pIC50 = 8.0, n = 4) and in other human ocular cells (IC50 = 15.8-31.6 nM, pIC50 = 7.5-7.8) and exhibited apparent noncompetitive antagonist properties in these cells, with an estimated dissociation constant (Kb) of 19.9 nM (pKb = 7.7, n = 6). This combination of mast cell mediator release inhibition and selective H1 receptor antagonism suggests that olopatadine may be particularly useful in the treatment of ocular allergic diseases. Indeed, olopatadine has recently shown clinical efficacy in an allergic conjunctivitis model in human subjects.

Anti-Allergic Agents↗
PubMed1996

Airway function correlates with circulating eosinophil, but not mast cell, markers of inflammation in childhood asthma.

BACKGROUND: Lung function tests, including forced expiratory volume in one second (FEV1), forced expiratory flow at 25-75% of vital capacity (FEF25-75%) and provocation concentrations of histamine which reduce FEV1 by 20% (PC20), are used as indicators of airway form and function in bronchial asthma. Recently, markers of eosinophil activation in bronchial lavage and serum have been suggested as a measure of eosinophil mediated inflammation in the airways. These include eosinophil cationic protein (ECP), eosinophil protein X (EPX) (also known as eosinophil derived neurotoxin) and eosinophil peroxidase (EPO). Similarly, serum tryptase has been used as a marker of mast cell activation in systemic anaphylaxis. OBJECTIVES: We measured both sets of indices in a group of children with moderately severe asthma to assess the contribution of eosinophil and mast cell mediated events to airflow limitation and bronchial hyperresponsiveness. METHODS: Forty-eight children aged 5-10 years had spirometric assessments, histamine challenges and blood sampling on the same occasion. After analysis of sera, the indices were compared. RESULTS: The eosinophil markers ECP and EPX correlated very well with each other. They showed a moderate negative correlation with PC20 for histamine. EPX was also found to negatively correlate with FEV1 and FEF25-75%. Serum tryptase levels showed no such correlates with airway function. CONCLUSION: These results suggest that serum markers of eosinophil activation correlate with airway function in childhood asthma, and may be of value in assessing the severity of the disease. It further supports the notion that childhood asthma has a similar immunopathology to that occurring in adults, with predominance of eosinophil mediated inflammation.

Asthma↗
PubMed1996

[Reration between symptom and counts of scattering Japanese cedar pollen--examination of clinical observation and repeated provocation].

We examined relation with the scattering number of Japanese cedar pollens in Mibu, Tochigi, Japan and a nasal allergy symptom expects from pollen observation at an appearance and symptom progress in detail. We investigated appearance day of nasal allergy symptom and relation with pollen number. High relation was admitted as accumulate rate of nasal allergy as accumulation pollen during a numerical square root. When it was long period of the first observation day of pollen to the first day of pollen scatterring, the rate of nasal allergy symptom became high. An equilateral correlation was admitted as symptom a score in symptom progress and relation with pollen number accumulation pollen during a numerical square root. ECP and tryptase value in nose juice washing liquid rose with progressive of symptom. Early phase reaction progresses in a continuation nose mucous membrane induction examination and ECP in nose juice washing and tryptase rose with it. Late phase reaction was halved with positive group by negative group, progress of reaction by repetition of induction was not accepted. In our conclusion, a symptom is caused by little pollen being exposed repeatedly and that a symptom will become worsen was observed by numerical accumulation pollen.

Blood Proteins↗
PubMed1995

Cellular activation products in osteoarthritis synovial fluid.

In order to address the issue of the role of mast cells (MC) and nitric oxide (NO) in rheumatic synovial-fluid diseases, synovial fluid (SF) collected from the knee of patients with osteoarthritis (OA), articular chondrocalcinosis (ACC) or rheumatoid arthritis (RA) was examined for the levels of mast cells (MC), histamine, tryptase, phospholipase A2 and nitrite. MC counts were found to be elevated in the SF of OA patients as compared with RA patients. Histamine content in SF parallelled the number of MC. Tryptase levels were elevated in OA in comparison to RA and ACC, but the difference was not statistically significant. Identical PLA2 levels were recorded among the 3 groups. Nitrite concentrations were also higher in SF from OA patients as compared to RA patients. These results suggest that mast cells (MC), in association with various inflammatory cells, may contribute to inflammation and cartilage breakdown in osteoarthritis (OA).

Aged↗
PubMed1996

Relationship between nasal hyperreactivity, mediators and eosinophils in patients with perennial allergic rhinitis and controls.

BACKGROUND: In perennial allergic rhinitis, patients are almost daily exposed to aeroallergens. This ongoing allergic reaction results in increased sensitivity to allergens and non-specific stimuli. It is generally known that inflammatory cells and mediators are involved in the pathogenesis of the allergic reaction. OBJECTIVES: To study the relationship between nasal hyperreactivity and nasal inflammation during natural allergen exposure. METHODS: In 48 patients with perennial allergic rhinitis and in 11 volunteers a nasal brush, a nasal lavage and a histamine challenge were performed. Nasal inflammation was estimated by the number of eosinophils, levels of albumin, tryptase, prostaglandin D2 (PGD2), eosinophil cationic protein (ECP) and leukotriene C4/D4/E4 (LTC4/D4/E4). RESULTS: In contrast to PGD2 and tryptase, eosinophils (1.9 vs 0%, P = 0.0023), LTC4/ D4/E4 (17.51 vs 1.43 pg/mL, P < 0.0001) and albumin (8.61 vs 2.37 mg/mL, P = 0.0008) were significantly increased in rhinitis patients as compared with controls. Patients also showed increased responses to nasal histamine challenge assessed using a composite symptom score (21.5 vs 4 points, P < 0.0001). The nasal response to histamine was weakly correlated with the total number of eosinophils in the cytospin (correlation coefficient r = 0.38, P = 0.009). CONCLUSION: Nasal hyperreactivity is correlated with the percentage of eosinophils in patients with perennial rhinitis. The patients' mediator profiles suggest that eosinophils are important in the ongoing allergic reaction and nasal hyperreactivity.

Adult↗
PubMed1996

Granulocyte proteins in serum in childhood asthma: relation to spirometry and therapy.

BACKGROUND: Measurement of markers of eosinophil activation in asthmatics provides information indicative of ongoing inflammatory processes in the airways. OBJECTIVES: This study was conducted to determine the correlations between serum markers of allergic inflammation with spirometry parameters in asthmatic children in different treatment groups. METHODS: Blood eosinophils, serum levels of eosinophil cationic protein (ECP), eosinophil protein X (EPX), myeloperoxidase (MPO) and tryptase were measured simultaneously with serial measurements of FEV1/FVC, FEF25-75 and FEF in 60 children with acute asthma on admission and after 2, 14, 30 and 60 days. Group A received bronchodilators only (n = 20), group B received sodium cromoglycate (SCG) (n = 20) and group C received oral and/or inhaled corticosteroids (n = 20). RESULTS: Oral steroid treatment (2 mg/kg/day), given at the onset of the asthma attack, resulted in significant reduction in the ECP and EPX levels in all the children. However, these reduced ECP and EPX levels were not sustained in the children, even in those who continued on maintenance steroid treatment. Significant, but inconsistent, correlations between ECP, EPX with total eosinophil count, Percentage eosinophils and spirometry parameters were observed at the different time-points. Tryptase levels were normal in all subjects. There were no significant correlations between myeloperoxidase levels and the spirometry parameters or eosinophil parameters. Serial monitoring of ECP and EPX levels was found to be of some use in predicting clinical outcome in certain steroid-dependent asthmatics (group C) but of no value in the mild asthmatics (group A). CONCLUSION: While elevation of ECP, EPX and MPO in the serum of childhood asthmatics suggests ongoing inflammation and may inversely correlate with spirometry parameters in some patients, the relationship between these markers and airway function is not a simple one.

Adolescent↗
PubMed1996

Mast cells in acute cellular rejection of human renal allografts.

Mast cells (MCs), few in the normal kidney, are found in increased number in the renal parenchyma in diseases associated with persistent chronic inflammation. MCs are not easily identified in routinely processed archival tissue sections with histochemical stains. A more reliable method of detection was provided with the introduction of MC tryptase-specific monoclonal antibodies. To determine the possible role of MCs in renal allograft rejection, we studied 28 biopsy specimens from renal allografts that had been in place for various lengths of time (from 3 days to 40 months) in patients whose primary diagnosis was acute interstitial rejection; the specimens were associated with varying degrees of interstitial fibrosis, edema, and hemorrhage. The specimens were graded on a semiquantitative scale (from 0 to 3+) for the severity of rejection, the degree of interstitial fibrosis, interstitial edema, and interstitial hemorrhage. Eosinophils, plasma cells, and MCs were quantitatively evaluated in these biopsy specimens. MCs were detected by use of a commercially available anti-MC tryptase monoclonal antibody, which proved to be an excellent tool to detect MCs in routinely processed paraffin sections. A positive correlation was found between the number of MCs and the time since transplantation (R = 0.841, P < 0.005) and between the number of MCs and the severity of interstitial fibrosis (R = 0.489, P < 0.005), as well as with interstitial edema (R = 0.517, P < 0.005). MCs were increased in number in patients with moderate (n = 18; mean, 18.00 MCs per 10 high power fields [HPFs]) and severe (n = 5; mean, 12.20 MCs per 10 HPFs) acute rejection compared with patients with mild (n = 5; mean, 2.44 MCs per 10 HPFs) acute rejection and normal kidneys (n = 6; mean, 1.75 MCs per 10 HPFs). These results suggested that MCs might play a role in the process of acute rejection of renal allografts and in the development of interstitial fibrosis.

Acute Disease↗
PubMedDate not supplied

Interactions of proteases and protease inhibitors in Sertoli-germ cell cocultures preceding the formation of specialized Sertoli-germ cell junctions in vitro.

The biochemical mechanism(s) by which germ cells can form specialized junctions with Sertoli cells in the seminiferous epithelium at various stages of the spermatogenic cycle is unknown. This study sought to examine the biochemical changes that are involved when germ cells are cocultured with Sertoli cells in vitro preceding the establishment of specialized Sertoli-germ cell junctions. While isolated germ cells were allowed to attach to Sertoli cells, media from both the apical and basal compartments of bicameral units were collected to assess serine and cysteine protease activity. The expression of selected serine and cysteine proteases and their corresponding inhibitors in these Sertoli-germ cell cocultures was also examined by RT-PCR. Using an [125I]-collagen film assay, a transient but significant increase in serine protease activity was noted in both the apical and basal compartments when germ cells began to settle onto the Sertoli cell monolayer preceding the formation of intercellular junctions. A specific tryptase (RNK-Tryp 2, a serine protease formerly cloned from a rat granular lymphocyte leukemia cell line, RNK-16, cDNA expression library) was shown to be expressed exclusively by Sertoli cells and not germ cells. Furthermore, Sertoli cell tryptase expression as well as urokinase plasminogen activator (u-PA, also a serine protease) increased significantly when germ cells were adhering to Sertoli cells. The decline in total serine protease activity when Sertoli-germ cell junctions were being formed was accompanied by a concomitant increase in alpha2-macroglobulin (alpha2-MG, a nonspecific protease inhibitor) expression. No significant changes in cysteine protease activity in either the apical or basal compartment were noted. However, there was a transient but significant increase in cathepsin L expression when germ cells were adhering to Sertoli cells preceding cell junction formation. The subsequent reduction in cathepsin L expression after this transient increase was accompanied by a concomitant increase in cystatin C expression. These results suggest that proteases and their corresponding inhibitors are working synergistically and are likely to be involved in the adherence of germ cells to Sertoli cells and the subsequent formation of intercellular junctions.

Animals↗
PubMed1998

Phenotypic and functional characterization of mast cells derived from renal tumor tissues.

Mast cells (MCs) originate from multipotent hematopoietic progenitor cells. However, MCs in various organs are heterogenous in terms of mediator or receptor expression and response to diverse stimuli. We characterized the phenotype and functional properties of human renal mast cells (HRMCs). Tissue was obtained from 17 patients suffering from renal tumors (transitional cell carcinoma, n = 4; renal cell carcinoma, n = 13). HRMCs were isolated by collagenase digestion. Double staining with toluidine blue and immunofluorescence using monoclonal antibodies (mAbs) revealed expression of stem cell factor (SCF)-receptor (c-kit/CD117), CD9, CD29, CD33, CD43, CD44, CD54, and CD63 on HRMCs. In contrast, HRMCs were not recognized by mAbs to CD2, CD3, CD4, CD11b, CD14, CD15, CD16, CDw17, CD19, or CD23. HRMCs were also negative for CD116 (granulocyte-macrophage colony-stimulating factor [GM-CSF] receptor alpha), CD123 (interleukin [IL]-3Ralpha), CD121a (IL-1R type I), CD122 (IL-2Rbeta), and CD127 (IL-7R) and were also found to lack C5aR (CD88). Ligand-induced activation of HRMCs through immunoglobulin (Ig)E-R or SCF-R (c-kit) resulted in histamine secretion (control: <10%; alphaIgE, 1 microg/mL: 50.12 +/-5.18%; rhSCF, 100 ng/mL: 29.24 +/- 22.39), whereas recombinant C5a, erythropoietin (EPO), IL-1 through 10, and GM-CSF exerted no effects. As determined by in situ staining, HRMCs contained tryptase, but only low or undetectable amounts of chymase. Electron microscopy confirmed the presence of MCs in renal tissues and revealed a scroll-rich granule population in HRMCs. Together, HRMCs are tryptase+, C5aR- mast cells exhibiting phenotypic and functional properties similar to those of lung MCs.

Adult↗
PubMed1997

Markers of airway inflammation in preschool wheezers.

Airways inflammation markers may help in predicting prognosis, in diagnosis and in monitoring respiratory diseases. Inflammation markers specific for certain cells may indicate the nature of the inflammatory processes, whilst others indicate stage and intensity. Intercellular adhesion molecule 1 (ICAM-1) helps to establish contact between the antigen-presenting cell and T-lymphocytes. Soluble serum ICAM-1 is increased in developing chronic lung disease of the newborn and atopic bronchial asthma. ICAM-1 is also the major human rhinovirus receptor. Interleukin 4 and interferon-gamma regulate the immunoglobulin E response, are difficult to measure in serum, and most groups employ stimulated cell cultures. These early inflammation markers may have predictive value. The leukotrienes are released from mast cells and eosinophils. Leukotriene (LT) B4 may be analysed in serum, whereas the cysteinyl leukotrienes, LTC4, LTD4, LTE4, may be assessed in urine. Serum LTB4 and urinary LTE4 have been found to be elevated during acute wheezy exacerbations. Tryptase is released from mast cells and is elevated in serum during acute anaphylaxis. However, tryptase has not been found to be related to inflammatory activity under other conditions. Myeloperoxidase is released from neutrophils, and serum levels are elevated in asthma, respiratory infections and other chronic lung diseases. The eosinophil markers eosinophil cationic protein (ECP) and eosinophil protein X (EPX) reflect eosinophil activation. ECP in serum and EPX in urine are elevated in asthma, atopic eczema and other conditions with eosinophil activation. They are related to symptom activity in asthma and atopic eczema and are influenced by anti-inflammatory therapy. In early wheezing, serum ECP may have predictive value. Serum ECP is dependent upon sampling procedures. Nitrogen oxide in exhaled air reflects inflammatory activity in asthma, and is influenced by anti-inflammatory therapy. However, in young children there are sampling difficulties.

Asthma↗
PubMed1998

Calcification in atherosclerotic plaque of human carotid arteries: associations with mast cells and macrophages.

Calcification has been examined in 250 samples of atherosclerotic lesions (types II to VI) of human carotid arteries using von Kossa and haematoxylin staining. Early calcification described as 'stippling' was first noted in stage III specimens, with intermediate and solid calcifications becoming increasingly prominent within advanced plaques, especially stages Vb and VI. Although the relative frequencies of stippling, intermediate and large calcified deposits varied between plaques of the same stage, the prevalent sites of calcification were recognized as the deeper regions of the intima and the atheroma. Immunolocalization and histochemical techniques were used to identify the associations of mast cells (MCs), macrophages, smooth muscle cells (SMCs), and elastin with the different stages of calcification. Early, dispersed stippling was commonly associated with local accumulations of macrophages (HAM56 and CD68-positive), MCs and extracellular MC tryptase, the presence of immunoreactive elastin, but the relative absence of SMCs. Intermediate stages of calcification described as 'morula' deposits were also associated with local increases in the numbers of macrophages and MCs. Larger calcified deposits, even within the same plaque specimen, showed no regular pattern of cellular or elastin associations. However, in the vast majority of specimens, macrophages represented the predominant cell type associated with different phases of calcification. By contrast, the calcification less frequently observed in the media beneath advanced plaques was commonly associated with SMCs and elastin; only rarely were macrophages or MCs present. These studies are the first to demonstrate that macrophages, MCs, and extracellular tryptase frequently occupy micro-environmental loci showing the first stages of calcification within the atherosclerotic plaque; similar associations with more advanced mineral deposits are discussed in relation to plaque rupture.

Aged↗
PubMed1995

Inflammatory markers in nasal lavage fluid from Industrial Arts teachers.

Exposure to wood dust can cause allergic and nonallergic rhinitis. Inflammatory markers [cells, albumin, tryptase, and eosinophil cationic protein (ECP)] were examined in nasal lavage fluid (NAL) sampled from 24 Industrial Arts (IA) teachers exposed to wood dust and other irritants and from 24 control subjects. The IA teachers had more nasal complaints but they did not differ significantly from the controls regarding ECP concentration (median 4.1 and 4.7 micrograms/L, respectively), albumin concentration (median 30.7 and 20.7 g/L), and percentage of neutrophils (median 56 and 34) in NAL. Tryptase was not detected. No marked inflammation was thus found, but the albumin concentration was higher in subjects reporting nasal stuffiness. In the IA teachers, a relationship between the percentage of neutrophils and the number of classes during the working week was found (Spearman's rank correlation coefficient 0.53, p < 0.01) indicating a possibility of wood-dust-related inflammatory effects on the nasal mucosa.

Albumins↗
PubMed1997

Mast cell responses in rheumatoid synovium. Association of the MCTC subset with matrix turnover and clinical progression.

OBJECTIVE: To determine the distribution of mast cell subsets and their density in synovium from normal subjects and from patients with osteoarthritis (OA) and rheumatoid arthritis (RA): METHODS: A sequential double-immunohistochemical staining technique was used to distinguish mast cells as positive for tryptase only (MCT) or for tryptase plus chymase (MCTC). Synovial tissue was obtained from RA patients (n = 16), OA patients (n = 18), and normal subjects (n = 15). Sections were analyzed to a depth of 1 mm from the synoviocyte lining layer by quantitative histomorphometry. Immunohistochemical data were correlated with histologic findings and clinical indices of disease activity. RESULTS: In normal synovium, the majority of mast cells belonged to the MCTC subset, outnumbering MCT cells by 5:1. The mean density of mast cells was significantly increased in RA synovia (60.9 cells/mm2) compared with OA (21.7 cells/mm2) and with normal (9.4 cells/mm2) synovia. Selective expansion of the MCT subset accounted for the increased mast cell density in OA. In RA, both subsets expanded and were associated with infiltrating inflammatory cells or with regions of highly cellular fibrous tissue (mainly MCTC). An association was noted between clinical parameters of activity or progression of rheumatoid disease and the density of MCTC cells, especially the density in the superficial layer of synovium. In RA synovia, we found no evidence of the chymase only, or MCC, immunophenotype. CONCLUSION: MCTC mast cells expand in RA but not OA, associate with regions of "active" fibrosis, and correlate with parameters of disease activity or progression of RA. These findings implicate the MCTC subset of mast cells in the pathologic mechanisms that mediate tissue damage in RA.

Arthritis, Rheumatoid↗
PubMed2002

Agonists of proteinase-activated receptor 2 induce TNF-alpha secretion from astrocytoma cells.

Proteinase-activated receptor 2 (PAR2) is cleaved and activated by trypsin or mast cell tryptase, and may play an important role in inflammation. We have investigated the potential of PAR2 agonists to modulate TNF-alpha secretion from human astrocytoma cell line CCF-STTG1. We found that CCF-STTG1 expresses PAR2 by RT-PCR and Western blot analysis. Agonists such as trypsin, the peptide SLIGKV-NH(2) (corresponding to the PAR2 tethered ligand), or mast cell tryptase directly signal to CCF-STTG1 to stimulate secretion of TNF-alpha but do not stimulate in the presence of soybean trypsin inhibitor (SBTI) or VKGILS-NH(2) (reverse peptide). The secretion of TNF-alpha by trypsin was significantly blocked by pretreatment with either 50 microM PD98059 or 1 microM SB203580. Furthermore, trypsin stimulated the activation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase homologue in CCF-STTG1 without any detectable activation of c-Jun N-terminal kinase (JNK). These results show that trypsin may induce TNF-alpha secretion following activation of ERK and p38 via PAR2 in CCF-STTG1.

Astrocytoma↗
PubMed2006

Imatinib mesylate in the treatment of systemic mastocytosis: a phase II trial.

BACKGROUND: Mastocytosis is characterized by the abnormal proliferation of mast cells in 1 or more organs. In most patients, a mutation is present in the gene for C-KIT, resulting in deregulation of the c-kit receptor. Imatinib mesylate is a potent inhibitor of c-kit receptor tyrosine kinase activity. Therefore, the authors evaluated the efficacy and safety of imatinib mesylate as treatment for patients with systemic mastocytosis. METHODS: Patients with systemic mastocytosis received imatinib mesylate orally at a dose of 400 mg once daily for 3 to 6 months. Low doses of prednisone were added during the first 2 weeks. Endpoints were reductions in serum tryptase, urinary N-methylhistamine excretion, skin lesions, the number of mast cells in bone marrow sections, hepatomegaly and/or splenomegaly, and symptoms. RESULTS: Of 14 patients who were included in the study, 11 patients had the D816V mutation. One patient expressed the FIP1L1-PDGFR-alpha rearrangement gene. In 2 patients, no mutation was found. In 10 patients, serum tryptase levels decreased >20%. In all patients, urinary N-methylhistamine excretion was reduced. In 8 of 13 evaluable patients, the number of mast cells in the bone marrow decreased. Skin symptoms diminished in 5 of 9 patients. Hepatosplenomegaly improved in 3 of 6 patients. Symptoms decreased in 8 of 13 patients. In all patients who had the D816V mutation, reductions in > or =2 endpoints were achieved. In the patient who expressed the FIP1L1-PDGFR-alpha rearrangement gene, a complete response was attained. In general, imatinib mesylate was tolerated well. CONCLUSIONS: Imatinib mesylate was effective in patients with systemic mastocytosis, including those who had the D816V mutation.

Adult↗
PubMed1992

Phenotypic characterization of KU812, a cell line identified as an immature human basophilic leukocyte.

The knowledge about the differentiation of basophilic leukocytes is fragmentary. This report discusses a detailed phenotypic characterization of molecular markers for hematopoietic differentiation in a basophilic leukemia cell line, KU812. The expression of markers for lymphoid, erythroid, neutrophil, eosinophil, monocytic, megakaryocytic, mast cell and basophil differentiation was analyzed at the mRNA level by Northern blots in the KU812 cells, and for reference, in a panel of human cell lines representative of the different hematopoietic differentiation lineages. KU812 was found to express a number of mast cell and basophil-related proteins, i.e. mast cell tryptase, mast cell carboxypeptidase A, high-affinity immunoglobulin (IgE) receptor alpha and gamma chains and the core protein for heparin and chondroitin sulphate synthesis. We found no expression of a number of monocyte/-macrophage or neutrophil leukocyte markers except for lysozyme. From earlier studies, it has been shown that lysozyme is not expressed in murine mucosal mast cell lines. This finding, together with the expression of the mast cell carboxypeptidase in KU812 might distinguish the phenotype of this cell line from that typical of mucosal mast cell lines in rodents. We found a low level of expression of the eosinophil and basophil marker, major basic protein, which might indicate a relationship between basophils and eosinophils. No expression is, however, detected with the eosinophil-specific markers eosinophil cationic protein, eosinophil-derived neurotoxin or eosinophil peroxidase. We also report an extensive screening for inducers of basophilic differentiation of the KU812 cells. The most efficient protocol of induction included serum starvation which led to a dramatic increase in a number of markers specific for mast cells and basophils such as tryptase, carboxypeptidase A and the heparin core protein. Finally, diisopropylfluorophosphate analysis of total protein extracts from KU812 show four labeled protein bands with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that this cell line expresses at least three previously undescribed serine proteases of which one or more could be a potential basophil-specific marker(s).

Basophils↗