scholarly journals Inactivation of thrombin by a complex between rat mast-cell protease 1 and heparin proteoglycan

1994 ◽  
Vol 299 (2) ◽  
pp. 507-513 ◽  
Author(s):  
G Pejler ◽  
K Söderström ◽  
A Karlström
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Binding Sites ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Macromolecular Complex ◽  
Mast Cell Protease ◽  
Peritoneal Mast Cells ◽  
Terminal Amino

Rat peritoneal mast cells were shown to inactivate thrombin rapidly. The thrombin-inactivating activity was purified to homogeneity by a combination of anion-exchange chromatography and h.p.l.c. on a Superdex 75 column. The purified thrombin inactivator had an apparent molecular mass of 29 kDa and an N-terminal amino acid sequence identical to rat mast-cell protease 1 (RMCP-1). After labelling of the mast cells in vivo with 35SO4(2-), RMCP-1 was recovered in a macromolecular complex with [35S]heparin proteoglycans. Dissociation of RMCP-1 from the heparin proteoglycans by Superdex 75 chromatography in the presence of 2 M NaCl resulted in a marked loss of the thrombin-inactivating activity displayed by the enzyme. When RMCP-1 was reconstituted with either endogenous [35S]heparin proteoglycans or standard pig mucosal heparin, the enzyme regained its thrombin-inactivating properties. Affinity chromatography of endogenous [35S]heparin on matrix-linked RMCP-1 demonstrated that all of the heparin molecules contained high-affinity binding sites for the mast-cell protease. In contrast, the endogenous mast-cell heparin showed low affinity for antithrombin, a protease inhibitor involved in the regulation of coagulation enzymes.

scholarly journals Biochemical and immunological characterization of multiple glycoforms of mouse mast cell protease 1: comparison with an isolated murine serosal mast cell protease (MMCP-4)

1993 ◽  
Vol 294 (1) ◽  
pp. 127-135 ◽  
Author(s):  
G F J Newlands ◽  
D P Knox ◽  
S R Pirie-Shepherd ◽  
H R P Miller
Keyword(s):  
Mast Cell ◽  
Amino Acid ◽  
Mast Cells ◽  
Western Blotting ◽  
Trichinella Spiralis ◽  
Double Diffusion ◽  
Terminal Amino Acid ◽  
Mast Cell Protease ◽  
Terminal Amino ◽  
Mouse Mast Cell

Five highly soluble, chymotrypsin-like, neutral serine proteases, with molecular masses in the range 30-33 kDa, were isolated from Trichinella spiralis-infected mouse small intestine. These enzymes were closely related antigenically on Western blotting and by Ouchterlony double diffusion using a polyclonal, cross-absorbed, sheep antibody raised against mouse mast cell protease-1 (MMCP-1) and on the basis of N-terminal amino acid sequence analysis, were identified as variant forms of MMCP-1. Substrate and inhibitor analysis confirmed that the five variants (MMCP-1 A-E) had similar characteristics, although highly significant (P = 0.025 to P < 0.0001) variations in Km and kcat, were detected. Against human alpha 1-proteinase inhibitor the Ki for MMCP-1C (45 pM) was significantly (P < 0.0001) greater than those for the other proteases (0.76-2.2 pM). The differences in electrophoretic mobility are probably a result of variable glycosylation, since removal of N-linked carbohydrate produced a polypeptide of approx. 28 kDa in each case which was, like the native enzyme, immunoreactive on Western blotting. A much less soluble 28 kDa enzyme was isolated from serosal mast cells and identified as MMCP-4 by N-terminal amino acid sequencing. Like MMCP-1 it has chymotrypsin-like substrate specificities with activity at neutral pH. However, it was antigenically distinct from MMCP-1 and, using sheep anti-MMCP-1, was not detected on Western blotting or by Ouchterlony double diffusion, e.l.i.s.a. or immunohistochemistry. This last technique established that the MMCP-1 variants were uniquely present in enteric mast cells, thereby providing a highly selective means of distinguishing the mucosal and connective tissue mast cell subsets in the mouse.

scholarly journals Multiple bidirectional alterations of phenotype and changes in proliferative potential during the in vitro and in vivo passage of clonal mast cell populations derived from mouse peritoneal mast cells

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 877-885 ◽  
Author(s):  
Y Kanakura ◽  
H Thompson ◽  
T Nakano ◽  
T Yamamura ◽  
H Asai ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tissue Type ◽  
Cell Populations ◽  
Proliferative Potential ◽  
Heparin Binding ◽  
Peritoneal Mast Cells ◽  
Proliferative Ability

Mouse peritoneal mast cells (PMC) express a connective tissue-type mast cell (CTMC) phenotype, including reactivity with the heparin-binding fluorescent dye berberine sulfate and incorporation of [35S] sulfate predominantly into heparin proteoglycans. When PMC purified to greater than 99% purity were cultured in methylcellulose with IL-3 and IL-4, approximately 25% of the PMC formed colonies, all of which contained both berberine sulfate-positive and berberine sulfate-negative mast cells. When these mast cells were transferred to suspension culture, they generated populations that were 100% berberine sulfate-negative, a characteristic similar to that of mucosal mast cells (MMC), and that synthesized predominantly chondroitin sulfate [35S] proteoglycans. When “MMC-like” cultured mast cells derived from WBB6F1-+/+ PMC were injected into the peritoneal cavities of mast cell-deficient WBB6F1- W/Wv mice, the adoptively transferred mast cell population became 100% berberine sulfate-positive. In methylcellulose culture, these “second generation PMC” formed clonal colonies containing both berberine sulfate-positive and berberine sulfate-negative cells, but exhibited significantly less proliferative ability than did normal +/+ PMC. Thus, clonal mast cell populations initially derived from single PMC exhibited multiple and bidirectional alterations between CTMC-like and MMC-like phenotypes. However, this process was associated with a progressive diminution of the mast cells' proliferative ability.

TLR3-induced activation of mast cells modulates CD8+ T-cell recruitment

Blood ◽  
2005 ◽  
Vol 106 (3) ◽  
pp. 978-987 ◽  
Author(s):  
Zane Orinska ◽  
Elena Bulanova ◽  
Vadim Budagian ◽  
Martin Metz ◽  
Marcus Maurer ◽  
...  
Keyword(s):  
Mast Cell ◽  
T Cell ◽  
Mast Cells ◽  
Cd8 T Cell ◽  
Poly I:C ◽  
Cell Recruitment ◽  
Polycytidylic Acid ◽  
Peritoneal Mast Cells

AbstractMast cells play an important role in host defense against various pathogens, but their role in viral infection has not been clarified in detail. dsRNA, synthesized by various types of viruses and mimicked by polyinosinic-polycytidylic acid (poly(I:C)) is recognized by Toll-like receptor 3 (TLR3). In this study, we demonstrate that poly(I:C) injection in vivo potently stimulates peritoneal mast cells to up-regulate a number of different costimulatory molecules. Therefore, we examined the expression and the functional significance of TLR3 activation in mast cells. Mast cells express TLR3 on the cell surface and intracellularly. After stimulation of mast cells with poly(I:C) and Newcastle disease virus (NDV), TLR3 is phosphorylated and the expression of key antiviral response cytokines (interferon β, ISG15) and chemokines (IP10, RANTES) is upregulated. Interestingly, mast cells activated via TLR3-poly(I:C) potently stimulate CD8+ T-cell recruitment. Indeed, mast-cell–deficient mice (KitW/KitW-v) given an intraperitoneal injection of poly(I:C) show a decreased CD8+ T-cell recruitment, whereas granulocytes normally migrate to the peritoneal cavity. Mast-cell reconstitution of KitW/KitW-v mice normalizes the CD8+ T-cell influx. Thus, mast cells stimulated through engagement of TLR3 are potent regulators of CD8+ T-cell activities in vitro and in vivo.

Transcriptional Regulation of a Distinct GATA-1 Isoform during Selection of the Mast and Erythroid Lineages.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1600-1600
Author(s):  
Clifford M. Takemoto ◽  
Amir H. Shahlaee ◽  
Ying Ye ◽  
Karen I. Zeller ◽  
Daniela Zablocki ◽  
...  
Keyword(s):  
Mast Cell ◽  
Transcription Factors ◽  
Cell Differentiation ◽  
Mast Cells ◽  
Binding Sites ◽  
Genomic Sequence ◽  
Erythroid Cells ◽  
Mrna Isoform ◽  
In Erythroid Cells

Abstract Current models of hematopoiesis suggest that in early, pluripotent progenitor cells, lineage-specific transcription factors are expressed at low levels. During differentiation, subsets of these transcription factors become dominantly expressed in a lineage-restricted fashion. Understanding how transcription factors are expressed in distinct cell-types is central to defining the regulatory events that occur during lineage selection. GATA-1 is an essential transcriptional regulator for the erythroid and megakaryocyte lineages, while it is absent in neutrophils and monocytes. PU.1, on the other hand, is a critical transcription factor for neutrophils and monocytes, but it is not abundantly expressed in erythroid cells. Although these two factors have been shown to be antagonistic in monocytic and erythroid cells, both GATA-1 and PU.1 are required for the normal development of the mast lineage (Migliaccio et al., 2003, Walsh et al., 2002). Here we show that mast cells express a unique mRNA isoform of GATA-1 that is distinct from the major erythroid/megakaryocyte isoform. It is related, but not identical to the Ib transcript that has been described as a minor expressed form in erythroid cells (Tsai et al., 1991) and as a major expressed form in RNA isolated from CFU-GM primary myeloid cultures (Seshasayee et al., 2000). This GATA-1 mast cell isoform (GATA-1mast) differs from the erythroid/megakaryocyte isoform by a unique, untranslated first exon that is alternatively spliced onto the downstream coding exons. In mast cells, GATA-1mast is expressed from a promoter separate from that utilized in megakaryocytic and erythroid cells. Comparative analysis of genomic sequence of the GATA-1 locus in this region reveals modules of extensive phylogenetic conservation in mammals, including stretches containing both highly conserved PU.1 and GATA binding sites. We have performed chromatin immunoprecipitation studies with GATA-1 antibodies and have defined multiple regions of in vivo binding within the GATA-1 locus in erythroid cells. Addtional studies are underway utilizing the Scanning ChIP procedure (Zeller et al., 2001) to determine in vivo GATA-1, GATA-2, and PU.1 binding sites of these factors to the GATA-1 locus in mast cells. In order to determine whether PU.1 positively regulates the expression of the mast cell GATA-1 isoform, we have examined GATA-1mast expression in PU.1 −/ − cells. PU.1 −/ − fetal liver cells cannot differentiate into mast cells in vitro; reintroduction of PU.1 expression restores mast cell differentiation. We show that PU.1 −/ − cells are deficient in expression of the GATA-1 mast cell mRNA isoform, and reintroduction of PU.1 into the PU.1 deficient cells markedly up-regulates the expression of GATA-1mast. Our findings demonstrate that PU.1 positively regulates a distinct GATA-1 isoform during mast cell differentiation. We propose a model in which GATA factors cooperate with PU.1 to direct cell-specific isoforms of transcriptional regulators during hematopoietic development.

scholarly journals Mast cell clones: a model for the analysis of cellular maturation.

1982 ◽  
Vol 95 (2) ◽  
pp. 435-444 ◽  
Author(s):  
S J Galli ◽  
A M Dvorak ◽  
J A Marcum ◽  
T Ishizaka ◽  
G Nabel ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Cytoplasmic Granule ◽  
Membrane Receptors ◽  
Cell Clones ◽  
Peritoneal Mast Cells ◽  
Immature Mouse ◽  
Plasma Membrane Receptors ◽  
And Function

Cloned mouse mast cells resemble, by ultrastructure, immature mast cells observed in vivo. These mast cell clones can be grown in the absence of any other cells, facilitating direct investigations of their biochemistry and function. We find that cloned mast cells express plasma membrane receptors (Fc epsilon R) that bind mouse IgE with an equilibrium constant (KA) similar to that of normal mouse peritoneal mast cells. In addition, cloned mast cells do not display detectable la antigens and cannot enhance lg secretion when added to lymphocyte cultures or mediate natural killer lysis. In the presence of 1 mM sodium butyrate, cloned mast cells stop dividing and acquire abundant electron-dense cytoplasmic granules similar to those of mature mast cells. Their histamine content increases concomitant with cytoplasmic granule maturation and may exceed that of untreated mast cells by 50-fold. Unlike peritoneal mast cells, cloned mast cells incorporate 35SO4 into chondroitin sulfates rather than heparin. These findings demonstrate that, unlike fully differentiated mouse peritoneal mast cells, cloned immature mouse mast cells contain no heparin and low levels of histamine. In addition, they establish that high-affinity Fc epsilon R are expressed early in mast cell maturation, well before completion of cytoplasmic granule synthesis and mediator storage.

scholarly journals Multiple bidirectional alterations of phenotype and changes in proliferative potential during the in vitro and in vivo passage of clonal mast cell populations derived from mouse peritoneal mast cells

Blood ◽  
1988 ◽  
Vol 72 (3) ◽  
pp. 877-885 ◽  
Author(s):  
Y Kanakura ◽  
H Thompson ◽  
T Nakano ◽  
T Yamamura ◽  
H Asai ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Tissue Type ◽  
Cell Populations ◽  
Proliferative Potential ◽  
Heparin Binding ◽  
Peritoneal Mast Cells ◽  
Proliferative Ability

Abstract Mouse peritoneal mast cells (PMC) express a connective tissue-type mast cell (CTMC) phenotype, including reactivity with the heparin-binding fluorescent dye berberine sulfate and incorporation of [35S] sulfate predominantly into heparin proteoglycans. When PMC purified to greater than 99% purity were cultured in methylcellulose with IL-3 and IL-4, approximately 25% of the PMC formed colonies, all of which contained both berberine sulfate-positive and berberine sulfate-negative mast cells. When these mast cells were transferred to suspension culture, they generated populations that were 100% berberine sulfate-negative, a characteristic similar to that of mucosal mast cells (MMC), and that synthesized predominantly chondroitin sulfate [35S] proteoglycans. When “MMC-like” cultured mast cells derived from WBB6F1-+/+ PMC were injected into the peritoneal cavities of mast cell-deficient WBB6F1- W/Wv mice, the adoptively transferred mast cell population became 100% berberine sulfate-positive. In methylcellulose culture, these “second generation PMC” formed clonal colonies containing both berberine sulfate-positive and berberine sulfate-negative cells, but exhibited significantly less proliferative ability than did normal +/+ PMC. Thus, clonal mast cell populations initially derived from single PMC exhibited multiple and bidirectional alterations between CTMC-like and MMC-like phenotypes. However, this process was associated with a progressive diminution of the mast cells' proliferative ability.

scholarly journals Mouse bone marrow-derived mast cells (mBMMC) obtained in vitro from mice that are mast cell-deficient in vivo express the same panel of granule proteases as mBMMC and serosal mast cells from their normal littermates.

1994 ◽  
Vol 180 (1) ◽  
pp. 67-73 ◽  
Author(s):  
K K Eklund ◽  
N Ghildyal ◽  
K F Austen ◽  
D S Friend ◽  
V Schiller ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cell ◽  
Steady State ◽  
Mast Cells ◽  
Induced Expression ◽  
Mast Cell Protease ◽  
Steady State Levels ◽  
Mouse Mast Cell

The ear, skin, and purified serosal mast cells of WBB6F1/J-(+/+) (WB-(+/+)) and WCB6F1/J-(+/+) (WC-(+/+)) mice contain high steady-state levels of the transcripts that encode mouse mast cell protease (mMCP) 2, mMCP-4, mMCP-5, mMCP-6, and mouse mast cell carboxypeptidase A (mMC-CPA). In contrast, no mast cell protease transcripts are present in abundance in the ear and skin of WBB6F1/J-W/Wv (W/Wv) and WCB6F1/J-Sl/Sld (Sl/Sld) mice which are mast cell-deficient in vivo due to defects in their c-kit and c-kit ligand genes, respectively. We now report that the immature bone marrow-derived mast cells (mBMMC) obtained in vitro with recombinant interleukin 3 (rIL-3) or WEHI-3 cell conditioned medium from WB-(+/+), WC-(+/+), W/Wv, and Sl/Sld mice all contain high steady-state levels of the mMCP-2, mMCP-4, mMCP-5, mMCP-6, and mMC-CPA transcripts. As assessed immunohistochemically, mMCP-2 protein and mMCP-5 protein are also present in the granules of mBMMC from WB-(+/+), WC-(+/+), and W/Wv mice. That Sl/Sld and W/Wv mBMMC contain high steady-state levels of five granule protease transcripts expressed by the mature serosal, ear, and skin mast cells of their normal +/+ littermates suggests that c-kit-mediated signal transduction is not essential for inducing transcription of these protease genes. Because rIL-4 inhibits the rIL-10-induced expression of mMCP-1 and mMCP-2 in BALB/cJ mBMMC, the ability of rIL-4 to influence protease mRNA levels in WC-(+/+) mBMMC and W/Wv mBMMC was investigated. Although rIL-10 induced expression of the mMCP-1 transcript in WC-(+/+) and W/Wv mBMMC, rIL-4 was not able to suppress the steady-state levels of the mMCP-1 transcript or any other protease transcript in these cultured mast cells. Thus, not only do BALB/cJ mBMMC express fewer granule proteases than mBMMC from mast cell-deficient strains and their normal littermates but the subsequent induction of late-expressed proteases in BALB/cJ mBMMC is more tightly regulated by IL-3 and IL-4.

Abstract 471: Systemic Chymase Inhibition Directs Atherosclerotic Plaques Towards a Stable Phenotype in ApoE Deficient Mice

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ilze Bot ◽  
Sandra H van Heiningen ◽  
Jurgen Fingerle ◽  
Hans Hilpert ◽  
Theo van Berkel ◽  
...  
Keyword(s):  
Mast Cell ◽  
Mast Cells ◽  
Cell Activation ◽  
Control Diet ◽  
Mast Cell Activation ◽  
Human Coronary Artery ◽  
Plaque Stability ◽  
Peritoneal Mast Cells ◽  
Chymase Inhibitor

Activated mast cells have been identified at the site of rupture in human coronary artery plaques and appear to contribute considerably to plaque progression and stability. We and others have previously demonstrated that the mast cell constituents chymase and tryptase promote apoptosis of plaque cells. In this study, we aimed to investigate whether inhibition of mast cell chymase by a specific chymase inhibitor indeed has a beneficial effect on plaque stability. Preincubation of 48/80 activated MC/9 murine mast cells or freshly isolated peritoneal mast cells with chymase inhibitor RO5010226 – 000 – 004 (RO501; 1 μM) inhibited mast cell activation, as illustrated by a decreased β-hexosaminidase activity in the releasate (−41% compared to control MC/9 cells, *P=0.04, and −80% compared to control peritoneal mast cells, *P=0.02) as well as chymase release and activity (−71% and −65%, *P=0.04, respectively). Next, we addressed whether chymase inhibition also was effective in vivo. Atherosclerotic carotid artery lesions were induced in ApoE −/− mice by perivascular collar placement; during lesion development mast cells were activated by a DNP challenge once weekly for 4 weeks. Concomitantly, a subset of mice received the chymase inhibitor (50 mg/kg/day, n=14) as diet supplement, leading to continuous serum concentrations of ~2 μM or control diet (n=12). After 6 weeks, the advanced plaques were analyzed for size and stability. While plaque size did not differ, collagen content of the lesions was 2-fold enhanced in mice treated with the chymase inhibitor compared to controls (RO501: 1.4 ± 0.5% versus controls: 0.7 ± 0.2%). This was accompanied by a significant decrease in necrotic core size of the plaques (RO501: 52 ± 3% versus controls: 41 ± 4%, *P=0.04) as well as by an increased plaque cellularity (RO501: 2.6 ± 0.1*10 3 versus controls: 2.3 ± 0.1*10 3 cells/mm 2 tissue). In agreement with these data we did observe increased peritoneal leukocyte numbers in the RO501 treated mice (RO501: 4.2 ± 1.1*10 6 cells versus 2.2 ± 0.3*10 6 cells in controls, *P=0.04). In conclusion, our data suggest that chymase inhibition indeed results in enhanced plaque stability, identifying chymase inhibition as a new therapeutic approach in the prevention of acute coronary syndromes.

scholarly journals Loss of Histochemical Identity in Mast Cells Lacking Carboxypeptidase A

2005 ◽  
Vol 25 (14) ◽  
pp. 6199-6210 ◽  
Author(s):  
Thorsten B. Feyerabend ◽  
Heinz Hausser ◽  
Annette Tietz ◽  
Carmen Blum ◽  
Lars Hellman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mast Cell ◽  
Mast Cells ◽  
Alcian Blue ◽  
Cell Phenotype ◽  
Carboxypeptidase A ◽  
Forward Scatter ◽  
Peritoneal Mast Cells

ABSTRACT Mast cell carboxypeptidase A (Mc-cpa) is a highly conserved secretory granule protease. The onset of expression in mast cell progenitors and lineage specificity suggest an important role for Mc-cpa in mast cells. To address the function of Mc-cpa, we generated Mc-cpa-null mice. Mc-cpa − / − mast cells lacked carboxypeptidase activity, revealing that Mc-cpa is a nonredundant enzyme. While Mc-cpa − / − peritoneal mast cells were ultrastructurally normal and synthesized normal amounts of heparin, they displayed striking histochemical and biochemical hallmarks of immature mast cells. Wild-type peritoneal mast cells had a mature phenotype characterized by differential histochemical staining with proteoglycan-reactive dyes (cells do not stain with alcian blue but stain with safranin and with berberine) and a high side scatter to forward scatter ratio by flow cytometry and were detergent resistant. In contrast, Mc-cpa − / − peritoneal mast cells, like immature bone marrow-derived cultured mast cells, stained with alcian blue normally or weakly and either did not stain with safranin and berberine or stained weakly, had a low side scatter to forward scatter ratio, and were detergent sensitive. This phenotype was partially ameliorated with age. Thus, histochemistry and flow cytometry, commonly used to measure mast cell maturation, deviated from morphology in Mc-cpa − / − mice. The Mc-cpa − / − mast cell phenotype was not associated with defects in degranulation in vitro or passive cutaneous anaphylaxis in vivo. Collectively, Mc-cpa plays a crucial role for the generation of phenotypically mature mast cells.

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