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PubMed1988

Establishment of an immature mast cell line from a patient with mast cell leukemia.

A cell line showing many characteristics of immature mast cells has been established from the peripheral blood of a patient with mast cell leukemia. Cultured cells contain low levels of histamine, are stained metachromatically by toluidine blue, and contain chloroacetate esterase, aminocaproate esterase and tryptase activities. The cells lack T and B lymphocyte, as well as myeloid cell markers, and do not possess IgE receptors. Solid tumors of metachromatically positive cells have been successfully induced and serially passed in nude mice using 5-azacytidine transformed cells. This cell line may be useful for future studies of mast cells and their constituents.

Animals↗
PubMed1992

Sulfonyl fluoride serine protease inhibitors inactivate RNK-16 lymphocyte granule proteases and reduce lysis by granule extracts and perforin.

Cytolytic granules purified from natural killer lymphocytes (NK) contain a pore-forming protein (perforin) and a number of serine proteases. When these proteases are inhibited by serine protease-specific isocoumarin reagents the serine proteases are inactivated and the cytolytic activity of the granules is decreased. Paradoxically, it has been found that the general serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) frequently cannot block killing even though it inhibits many of the serine proteases. At the same time it has been reported that "purified" perforin alone can lyze cells. To address these inconsistencies we first compared the ability of PMSF and four new sulfonyl fluoride serine protease inhibitors to inhibit proteases and cell lysis. We determined the effects on lysis and the second order inhibition rate constants for five granule protease activities: ly-tryptase, ly-chymase, Met-ase (methionine cleaving), Ser-ase (serine cleaving) and Asp-ase (aspartic acid cleaving). One compound, 2-(Z-NH(CH2)2CONH)C6SO2F, was a potent inhibitor of Met-ase activity (k(obsd)/[I] = 162 M-1 s-1), ly-chymase activity (k(obsd)/[I] = 147 M-1 s-1), and granule-mediated as well as perforin-mediated lysis. PMSF was a poor inhibitor of granule proteases (k(obsd)/[I]'s less than 7 M-1 s-1 for four activities and no inhibition of Ser-ase); the lack of reactivity is consistent with the failure of PMSF to block granule lytic activity. We also prepared enriched perforin by anion exchange chromatography and showed that a ly-chymase and a Met-ase associated with perforin. By inhibiting these proteases we also inhibited lytic activity.

Animals↗
PubMedDate not supplied

Characterization, application and potential uses of biotin-tagged inhibitors for lymphocyte serine proteases (granzymes).

Cytotoxic lymphocytes and natural killer cells are able to kill their target cells in minutes. The death of the target cell occurs after the release of cytoplasmic granules from the effector cell. These granules contain the pore-forming protein perforin and serine proteases (granzymes). To date 10 genes encoding lymphocyte granzymes have been discovered; of these only four have been purified and characterized for their substrate specificity. Several are predicted to have a common chymase, like specificity which is found in the granule extracts. Others may need to be enriched as active enzymes before they can be evaluated for substrate hydrolysis. Due to the limitations of detection by substrate hydrolysis, a more sensitive method for the detection of dilute granules was needed. We report the differing reactivities of seven biotin (Bi)-tagged isocoumarin (IC) inhibitors for Asp-ase, chymase, tryptase and Met-ase granzymes. The inhibitors contained different substituents at their no. 3 position: methoxy (OMe), ethoxy (OEt), propoxy (OPr) or 2-phenylethoxy (OEtPh) groups. The OMe group conferred general reactivity, whereas the OEtPh group conferred selective reactivity with chymase granzymes. The inhibitors that contained the longest aminocaproyl (Aca) spacers between the biotin-tag and the isocoumarin ring mediated the most stable granzyme inactivation. These inhibitors were the most effective at blocking lysis of red blood cells by the granule extracts. The inhibitors were used in protein blotting experiments where the biotin was detected with an avidin-enzyme complex. Over 10 granzymes were labelled by the inhibitor Bi-Aca-Aca-IC-OMe. The inhibitors detected granzymes when they were not readily detected by substrate hydrolysis.

Amino Acid Sequence↗
PubMed1990

A trypsin-like proteinase appearing at 17 h and 17 min in the cell cycle time of HeLa cells correlates with the onset of DNA synthesis.

A trypsin-like proteinase appearing sharply at 17 h 17 min (17:17) in the cell cycle time of the synchronized and growing HeLa cells correlates with the onset of DNA synthesis, and the inhibition of the proteinase activity by trans-4-guanidinomethylcyclohexanecarboxylic acid 4-tert-butylphenyl ester (GMCHA-OPhBut) results in a 3 h retardation of the onset of the DNA synthesis. The proteinase activity is cell density dependent and completely inhibited by 100 microM GMCHA-OPhBut. The proteinase was named HeLa tryptase 17:17. The fact that the DNA synthesis occurred after the 3 h retardation in the presence of GMCHA-OPhBut strongly suggests the involvement of the alternative trigger for DNA synthesis in HeLa cells.

Cell Cycle↗
PubMedDate not supplied

Human mast cell proteases hydrolyze neurotensin, kinetensin and Leu5-enkephalin.

Purified mast cell carboxypeptidase cleaved the C-terminal leucines from Leu5-enkephalin (Leu-ENK), neurotensin (NT), and kinetensin (KT), with Km values of 36, 16, and 15 microM, and kcat values of 44, 51, and 53 s-1, respectively. To better predict potential in vivo hydrolysis products generated by mast cell proteases, these peptides were incubated with released skin mast cell supernatants. Leu5-enkephalin was hydrolyzed only by carboxypeptidase. Kinetensin was cleaved by tryptase, chymase, and carboxypeptidase to yield KT(1-3), KT(1-7), KT(1-8), KT(4-7), and KT(4-8), the last two peptides by the concerted action of two of the proteases. NT(1-11) and NT(1-12) were generated from neurotensin by chymase and carboxypeptidase, respectively.

Amino Acid Sequence↗
PubMed1995

Intrahepatic mast cells in chronic liver diseases.

Mast cells are known to be present in human liver but their distribution and density in normal livers and in chronic liver diseases have not previously been examined. In this study, we quantified mast cell numbers and examined their distribution in percutaneous biopsy specimens from normal livers (n = 8) and in two chronic progressive liver diseases: primary biliary cirrhosis (PBC) (n = 40) and alcoholic liver disease (n = 33). We compared differences in mast cell density between these two forms of chronic liver disease because it had been suggested that mast cells may play a role in the development of liver fibrosis, particularly in patients with chronic cholestatic liver disease who frequently have increased plasma histamine levels. Mast cells were identified by immunohistochemistry using a specific monoclonal antibody (AA1) raised against mast cell tryptase after an initial study showed this to be more sensitive for the detection of mast cells than the conventional histochemical stain, toluidine blue. Our results showed that small numbers of mast cells (3.9 +/- 3.3/mm2) are present within the portal tracts and sinusoids of normal livers. In progressive chronic liver disease, increased numbers of mast cells were present, which correlated with the increasing amounts of liver fibrosis present. We found significantly more mast cells in the PBC group compared with the alcoholic group for a given amount of fibrosis. Our findings suggest that mast cells and their mediators may play a role in liver fibrogenesis.

Biopsy↗
PubMed1984

Circulating proalbumin associated with a variant proteinase inhibitor.

The unique finding of normal proalbumin in human plasma provides an insight into the mechanism of propeptide cleavage. Proalbumin, present as 1-5% of the total albumin, was found in a boy whose prime problem was the presence of a mutant proteinase inhibitor, alpha 1-antitrypsin Pittsburgh (358 Met----Arg) [2]. The inferred structure of human proalbumin was confirmed as Arg-Gly-Val-Phe-Arg-Arg-Alb. On incubation with various enzymes (trypsin, tryptase, thrombin, chymotrypsin, chymase and cathepsin B), only trypsin was capable of converting proalbumin to albumin. There was no conversion when proalbumin was incubated with whole blood, plasma or serum. However, intravenous injection of proalbumin into a rat resulted in complete conversion to albumin, the half-life of this process being 6 h. We conclude that propeptide cleavage is dependent on a serine proteinase which is inhibited intracellularly, by the mutant inhibitor, and that all the albumin in the boy was secreted as proalbumin, but was subjected to a separate cleavage process after export from the hepatocyte.

Adolescent↗
PubMed1988

Localization, implications for function, and gene expression of chymotrypsin-like proteinases of cytotoxic RNK-16 lymphocytes.

Rat RNK-16 leukemia cells kill YAC-1, which are the cells lysed by rodent natural killer lymphocytes. We found chymotrypsin-like proteinase ('chymase') activity in the RNK-16 dense granules that also contain cytolytic activity. The chymase activity hydrolyzed the thiobenzyl peptide substrate Suc-Phe-Leu-Phe-SBzl and, in comparison to RNK-16 tryptase activity, was selectively inhibited by three different types of serine proteinase inhibitors. The selective inhibitors were the fungal aldehyde chymostatin, the chloromethylketone Z-Gly-Leu-Phe-CH2Cl, and the mechanism-based or 'suicide' inhibitor 7-amino-4-chloro-3-(2-phenylethoxy)isocoumarin. These proteinase inhibitors also blocked RNK-16 granule-mediated cytolysis. Chymostatin, a reversible inhibitor, delayed granule-mediated cytolysis, whereas the irreversible chloromethylketone and isocoumarin proteinase inhibitors completely abrogated granule-mediated cytolysis. The two irreversible inhibitors displayed biphasic inhibition of the chymase activity, indicating that at least two chymases are present in the granules. By Northern blot analysis, we found that RNK-16 mRNA hybridized strongly with a cDNA probe of CCPI, a mouse cytotoxic T lymphocyte serine proteinase gene. These data imply that chymase activity in the cytotoxic granules is important for cytolytic function and is likely to belong to a new subfamily of serine proteinases.

Animals↗
PubMed1989

Human mast cell proteases and mast cell heterogeneity.

Mast cell neutral proteases are distinctive markers of the MC(T) and MC(TC) cells in humans. Measurements of tryptase levels in vivo serve as an overall indicator of mast cell activity. Further research is needed to evaluate the functional role of these proteases as well as each mast cell type in situations related to both health and disease.

Biomarkers↗
PubMed2005

The faces of mast cell disease: bone marrow infiltrates in 3 patients with systemic mastocytosis.

The clinical spectrum of mast cell disease ranges from relatively innocuous and histologically subtle urticarial skin lesions to an aggressive and fatal leukemic form of mast cell proliferation. Not surprisingly, mast cell infiltrates may show significant microscopic heterogeneity, particularly in the bone marrow, the most common site of involvement in systemic mastocytosis (SM). Herein, 3 cases are presented to illustrate the clinical and morphologic heterogeneity of mast cell disease: the first patient, with long standing urticaria pigmentosa, developed anemia and thrombocytopenia; the second patient presented with a pathologic fracture; and the third patient was suspected to have refractory anemia. Upon bone marrow examination, all 3 patients showed mast cell infiltration with distinct morphologic features and all met the WHO criteria for aggressive systemic mastocytosis. Histochemical methods continue to play a role in the identification of mast cells, with some limitations depending on the degree of differentiation of the mast cells and tissue processing methods. Immunohistochemistry has contributed to the identification of mast cells. Coexpression of CD117 and CD25, as well as expression of the more specific immunohistochemical marker tryptase, is seen in systemic SM. The latter may also be employed as a serum marker in the diagnosis and follow-up of patients with SM. The mast cells, in the majority adults with SM, have somatic point mutations of KIT.

Aged↗
PubMed2006

Mast cell proteases: physiological tools to study functional significance of high density lipoproteins in the initiation of reverse cholesterol transport.

The extracellular fluid of the intima is rich in lipid-poor species of high density lipoproteins (HDL) that promote efficient efflux of cholesterol from macrophages. Yet, during atherogenesis, cholesterol accumulates in macrophages, and foam cells are formed. We have studied proteolytic modification of HDL by mast cell proteases as a potential mechanism of reduced cholesterol efflux from foam cells. Mast cells are present in human atherosclerotic lesions and, when activated, they expel cytoplasmic granules that are filled with heparin proteoglycans and two neutral proteases, chymase and tryptase. Both proteases were found to specifically deplete in vitro the apoA-I-containing prebeta-migrating HDL (prebeta-HDL) and other lipid-poor HDL particles that contain only apoA-IV or apoE. These losses led to inhibition of the high-affinity component of cholesterol efflux from macrophage foam cells facilitated by the ATP-binding cassette transporter A1 (ABCA1). In contrast, the diffusional component of efflux promoted by alpha-HDL particles was not changed after proteolysis. Mast cell proteases are providing new insights into the role of extracellular proteolysis of HDL as an inhibiting principle of the initial steps of reverse cholesterol transport in the atherosclerotic intima, where many types of protease-secreting cells are present.

Animals↗
PubMed2006

Transcriptional regulation of mouse mast cell protease-7 by TGF-beta.

Mouse mast cell protease-7 (mmcp-7) is a tryptase predominantly expressed in differentiated connective tissue-type mast cells. Previous study revealed that transforming growth factor-beta (TGF-beta) increases gene transcript of mmcp-7 in mast cells. The present study explored molecular mechanism of the up-regulation of mmcp-7 by TGF-beta. Luciferase-based reporter assays using deletion and point mutations of mmcp-7 promoter showed a critical region spanning nt -126 to -122 relative to the transcriptional start site, a Smad-binding element, for transcriptional activation by the TGF-beta pathway. In addition, a region from nt -104 to -98, a TPA-responsive element, was also necessary for the transactivation. Consistent with the current model for the TGF-beta signaling, Smad4 was required for the transcription of mmcp-7 by Smad3, a signal mediator of TGF-beta. Treatment with TGF-beta in mast cells resulted in the differential gene induction of the AP-1 components, i.e., transient induction of c-fos but not of c-jun and junB. Expression of c-fos further enhanced Smad3 and Smad4-induced transcription of mmcp-7, whereas c-jun expression inhibited the transcription. Our results suggest that TGF-beta stimulates mmcp-7 transcription through the Smad3-Smad4 pathway as well as c-fos induction, and that the AP-1 components distinctly related with the TGF-beta pathway.

Animals↗
PubMed2005

Expression of mRNA for four subtypes of the proteinase-activated receptor in rat dorsal root ganglia.

Proteinase-activated receptors (PARs) are members of the superfamily of G-protein coupled receptors that initiate intracellular signaling by the proteolytic activity of extracellular serine proteases. Three member of this family (PAR-1, PAR-3, and PAR-4) are considered thrombin receptors, whereas PAR-2 is activated by trypsin and tryptase. Recently, activation of PAR-2 signal was identified as a pro-inflammatory factor that mediates peripheral sensitization of nociceptors. Activation of PAR-1 in the periphery is also considered to be a neurogenic mediator of inflammation that is involved in peptide release. Here, we investigated the expression of these four members of PARs in the adult rat dorsal root ganglia (DRG) using radioisotope-labeled in situ hybridization histochemistry. We detected mRNA for all subtypes of PARs in the DRG. Histological analysis revealed the specific expression patterns of the PARs. PAR-1, PAR-2, and PAR-3 mRNA was expressed in 29.0+/-4.0%, 16.0+/-3.2%, and 40.9+/-1.3% of DRG neurons, respectively. In contrast, PAR-4 mRNA was mainly observed in non-neuronal cells. A double-labeling study of PARs with NF-200 and alpha calcitonin gene-related peptide (CGRP) also revealed the distinctive expression of PARs mRNA in myelinated or nociceptive neurons. This study shows the precise expression pattern of PARs mRNA in the DRG and indicates that the cells in DRG can receive modulation with different types of proteinase-activated receptors.

Animals↗
PubMed2005

Cardiac mast cell regulation of matrix metalloproteinase-related ventricular remodeling in chronic pressure or volume overload.

The chronic elevation in ventricular wall stress secondary to ventricular volume or pressure overload leads to structural remodeling of the muscular, vascular and extracellular matrix components of the myocardium. While initially a compensatory response, the progressive hypertrophy and ventricular dilatation induced by this condition ultimately have a detrimental effect on ventricular function, resulting in heart failure. Fibrillar collagen provides the skeletal framework which interconnects the cardiomyocytes, thereby maintaining ventricular shape and size and contributing to tissue stiffness. Accordingly, these myocardial collagen fibers must be disrupted for ventricular dilatation, sphericalization and wall thinning to occur. The presence of an abundant, latent matrix metalloproteinase (MMP) population which coexists with myocardial fibrillar collagen has been documented. Thus, the potential for collagen degradation to exceed synthesis exists should there be significant activation of this latent MMP system. Mast cells are known to store and release a variety of biologically active mediators including TNF-alpha and proteases such as tryptase and chymase, which can induce MMP activation. Increased cardiac mast cell density has been implicated in the pathophysiology of human end-stage cardiomyopathy and experimental myocardial infarction, hypertension and chronic volume overload secondary to mitral regurgitation and aorto-caval fistula. The potential role of cardiac mast cells in activating MMPs, which then results in fibrillar collagen degradation and adverse myocardial remodeling secondary to chronic volume and pressure overload will be the subject of this review.

Extracellular Matrix↗
PubMed2005

Chronic idiopathic urticaria: infiltrating cells and related cytokines in autologous serum-induced wheals.

The term chronic autoimmune urticaria (CAIU) is used for chronic urticaria in subjects who present a whealing response to the intradermal injection of autologous serum, suggesting the presence of pathogenic antibody activities. In this study, we examined 28 chronic urticaria subjects with positive autologous serum skin test (ASST), all of whom presented autologous serum-induced lesions at different evolutive stages. Punch biopsies were taken from lesional skin of six subjects at 10', eight subjects at 30', six subjects at 60', and four subjects each at 24 and 48 h. Immunological studies focussed on infiltrating cell immunophenotype and related cytokines, chemokines and chemokine receptors, adhesion molecules. Immunohistochemical staining was performed to measure expression of CD3, CD4, CD8, tryptase, eosinophil cationic protein, myeloperoxidase, basophil granular protein, IL-4, IL-5, IL-8, CCR3 and CXCR3, ICAM-1, VCAM and ELAM. Control staining was done on unaffected skin from the patients and normal skin from four healthy donors. The main infiltrating population was represented by neutrophils, seen focally in both unaffected skin (P = 0.001) and healthy controls (P = 0.003). IFN-gamma and IL-5 were expressed focally in autologous wheals. Significant staining for IL-4 was seen at 30'. CCR3 and CXCR3 were expressed less in autologous wheals than in uninvolved skin (P < 0.0001; P = 0.002). Cellular adhesion molecules (CAMs) reached their highest expression at 30' and 60' in induced lesions, and they showed strong expression also in unaffected skin (ICAM-1: P < 0.0001). Our data show that the immunoinflammatory features of ASST-induced wheals involve a prevalent role of lymphocytes (with a mixed Th1/Th2 response), with strong neutrophil infiltration and activity and involvement of the chemokine pathway. We interpreted the finding of inflammatory cells and mediator up-regulation in uninvolved CIU skin as a sign of prolonged and widespread "urticarial status".

Adult↗
PubMed2005

The hypereosinophilic syndromes: still more heterogeneity.

In 1968, the term hypereosinophilic syndromes (HES) was coined to refer to a spectrum of eosinophil-associated diseases presumed to be caused by an underlying immunological pathology. In the 1990s, the identification of an HES subset with T lymphocyte clonality and production of cytokines, particularly IL-5, validated this concept. Then, in 2002, imatinib mesylate, which was introduced for the treatment of chronic myelogenous leukemia, effectively controlled another subgroup of HES patients. Imatinib's target is a novel constitutively-active kinase. Most imatinib-responsive HES patients show an increased number of bone marrow mast cells and elevated serum tryptase; mast cells, lymphocytes and neutrophils express the novel kinase. This new information critically modifies our view of HES and indicates that several cell lines are altered and likely to contribute to HES pathophysiology.

Benzamides↗
PubMed2006

Targeting mast cells in the treatment of functional gastrointestinal disorders.

Enhanced knowledge of the pathophysiological basis of functional gastrointestinal disorders indicates that low-grade mucosal inflammation and mast cell hyperplasia are common findings. Mast cells are multipotent and mucosa-dwelling residents are uniquely located to communicate with host immune and nervous supersystems and with the gut microflora to provide tight microenvironmental conditions. Maintenance of homeostasis within this integrated defense system is crucial for symbiotic health, whereas breakdown of that balance might lead to uncontrolled mucosal and systemic inflammation. Numerous advances have recently emerged in the understanding of regulatory mechanisms of mast cell activation, development and homing to mucosal surfaces, as well as of the role of mast cells in key steps of mucosal inflammation. Such observations have stimulated the development of candidate drugs, such as tryptase or Syk inhibitors, that might be useful for the treatment of gastrointestinal functional disorders.

Gastrointestinal Diseases↗
PubMed2006

Aggressive systemic mastocytosis complicated by protein-losing enteropathy.

A 46-year-old woman was admitted to the hospital with complaints of chronic diarrhoea, vomiting and severe muscle weakness. Clinical examination showed a lethargic, malnourished, dehydrated patient with ascites and bilateral leg oedema. Laboratory evaluation revealed mild normochromic normocytic anaemia and severe hypoproteinaemia with hypoalbuminaemia. Upper gastrointestinal endoscopy showed a thickened, friable duodenal mucosa with multiple erosions. Colonoscopy revealed nodular, pseudopolypoid lesions with patchy erosions in the left hemicolon. Haematoxylin-eosin stained sections from biopsies of endoscopically abnormal bowel segments showed multi-focal aggregates of large, histiocyte-like cells with abundant pale cytoplasm in the lamina propria. These cells were negative on PAS, Ziehl-Neelsen, Giemsa and toluidine blue stains. Their immunophenotype was CD68 (+), c-kit/CD117 (+) and mast cell tryptase (+), which is consistent with mast cells. A trephine biopsy showed diffuse replacement of the bone marrow by atypical, monomorphic, frequently spindle-shaped mast cells. No associated haematopoietic malignancy was detected. The final diagnosis was aggressive systemic mastocytosis with involvement of the gastrointestinal tract complicated by protein-losing enteropathy. This association has not been reported previously. The patient has been treated with prednisolone and interferon-alpha and has since recovered.

Anti-Inflammatory Agents↗