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PubMed1991

Mast cell hyperplasia in atopic keratoconjunctivitis. An immunohistochemical study.

Immunohistochemical staining of conjunctival biopsies from four normal subjects and five patients with atopic keratoconjunctivitis (AKC) was performed, using a monoclonal antibody to human mast cell tryptase. A massive hyperplasia of mast cells in the patients with AKC was demonstrated. The mean mast cell density in the normal patients was 55.6/mm2 and in the patients with AKC, 172/mm2. The appearance of large numbers of mast cells in the conjunctival epithelium of AKC patients was also demonstrated. The possible pathophysiological significance of these findings is discussed.

Adolescent↗
PubMed2003

Mast cell-derived tumor necrosis factor induces hypertrophy of draining lymph nodes during infection.

Palpable swelling of regional lymph nodes is a common sequela of microbial infections but the mechanism responsible for the sequestration and subsequent coordination of lymphocyte responses within these dynamic structures remains poorly understood. Here we show that draining lymph nodes of mast cell-deficient mice did not demonstrate swelling after intradermal bacterial challenge. Testing of individual mast cell-derived products in this model indicated that tumor necrosis factor was the main mediator of nodal hypertrophy, whereas tryptase and histamine had no effect. After peripheral mast cell activation, both tumor necrosis factor concentrations and the recruitment of circulating T cells were increased within draining nodes. These results show a critical function for peripheral mast cell-derived tumor necrosis factor in regulating the hypertrophy of draining lymph nodes during infection.

Animals↗
PubMed2002

Expression of cyclooxygenase-2 (COX-2) in tumour and stroma compartments in cervical cancer: clinical implications.

This study aims at investigating the relationship between cyclooxygenase-2 expression in tumour vs stroma inflammatory compartment and its possible clinical role. The study included 99 stage IB-IV cervical cancer patients: immunostaining of tumour tissue sections was performed with rabbit antiserum against cyclooxygenase-2. CD3, CD4, CD8, CD25, Mast Cell Tryptase monoclonal antibodies were used to characterise stroma inflammatory cells in nine cervical tumours. An inverse relation was found between cyclooxygenase-2 levels (cyclooxygenase-2 IDV) of tumour vs stroma compartment (r=-0.44, P<0.0001). The percentage of cases showing high tumour/stromal cyclooxygenase-2 IDV ratio was significantly higher in patients who did not respond to treatment (93.3%) with respect to patients with partial (60.5%), and complete (43.7%) response (P= 0.009). Cases with a high tumour/stroma cyclooxygenase-2 IDV ratio had a shorter overall survival rate than cases with a low tumour/stroma cyclooxygenase-2 IDV (P<0.0001). In the multivariate analysis advanced stage and the status of tumour/stroma cyclooxygenase-2 IDV ratio retained an independent negative prognostic role. The proportion of CD3(+), CD4(+), and CD25(+) cells was significantly lower in tumours with high tumour/stroma cyclooxygenase-2 IDV ratio, while a higher percentage of mast cells was detected in tumours showing high tumour/stroma cyclooxygenase-2 IDV ratio. Our study showed the usefulness of assessing cyclooxygenase-2 status both in tumour and stroma compartment in order to identify cervical cancer patients endowed with a very poor chance of response to neoadjuvant therapy and unfavourable prognosis.

Adult↗
PubMed1998

Human mast cell chymase induces the accumulation of neutrophils, eosinophils and other inflammatory cells in vivo.

The roles of chymase in acute allergic responses are not clear, despite the relative abundance of this serine proteinase in the secretory granules of human mast cells. We have isolated chymase to high purity from human skin tissue by heparin-agarose affinity chromatography and Sephacryl S-200 gel filtration procedures, and have investigated the ability of human mast cell chymase to stimulate cell accumulation following injection into laboratory animals. Injection of chymase provoked marked neutrophilia and eosinophilia in the skin of Dunkin Hartley guinea-pigs. Compared with saline injected control animals, there were some 60 fold more neutrophils and 12 fold more eosinophils present at the injection site. Following injection of chymase into the peritoneum of BALB/c mice, there were up to 700 fold more neutrophils. 21 fold more eosinophils, 19 fold more lymphocytes and 7 fold more macrophages recovered than from saline injected controls at 16 h. Doses of chymase as low as 5 ng (1.7 x 10(-13) mole) stimulated an inflammatory infiltrate, and significant neutrophilia was elicited within 3 h. The chymase induced cell accumulation in both the guinea-pig and mouse models was dependent on an intact catalytic site, being reduced by co-injection of proteinase inhibitors or heat inactivation of the enzyme. Co-injection of histamine or heparin significantly reduced the chymase induced neutrophil accumulation, whereas neither histamine nor heparin by themselves had any effect on the accumulation of nucleated cells. No synergistic or antagonist interactions between chymase and tryptase were observed when these two major mast cell proteinases were co-injected into the mouse peritoneum. Our findings suggest that chymase may provide an potent stimulus for inflammatory cell recruitment following mast cell activation.

Animals↗
PubMed2006

Regulation of IL-1-induced selective IL-6 release from human mast cells and inhibition by quercetin.

Mast cells are involved in allergic reactions, but also in innate immunity and inflammation. Crosslinkage of mast cell Fc immunoglobulin E receptors (FcvarepsilonRI) by multivalent antigen triggers secretion of granule-stored mediators, as well as de novo synthesis of cytokines, including interleukin (IL)-6. We showed recently that the proinflammatory cytokine IL-1 stimulates human leukemic mast cells (HMC-1) and human umbilical cord blood-derived cultured mast cells (hCBMCs) to release newly synthesized IL-6 without tryptase in the absence of degranulation. Here, we investigated several signal-transduction pathways activated by IL-1 leading to IL-6 production by HMC-1 and hCBMCs. We also investigated the effect of the flavonol quercetin that was recently shown to strongly inhibit IL-6 secretion in response to allergic stimulation from hCBMCs.IL-1 stimulated p38, but did not activate extracellular signal-regulated kinase (ERK) or c-jun N-terminal kinase (JNK); it also did not activate protein kinase C (PKC) isozymes alpha, beta, mu and zeta, except for PKC-theta, which was phosphorylated. The p38 inhibitor SB203580 and the PKC inhibitors Calphostin C and Gö6976 completely inhibited IL-1-induced IL-6 production. Quercetin 1-100 microM inhibited IL-1-induced IL-6 secretion, p38 and PKC-theta phosphorylation in a dose-dependent manner. These results indicate that IL-1-stimulated IL-6 production from human mast cells is regulated by biochemical pathways distinct from IgE-induced degranulation and that quercetin can block both IL-6 secretion and two key signal transduction steps involved.

Carbazoles↗
PubMed2006

Suppression of pancreatitis-related allodynia/hyperalgesia by proteinase-activated receptor-2 in mice.

1 Proteinase-activated receptor-2 (PAR2), a receptor activated by trypsin and tryptase, is abundantly expressed in the gastrointestinal tract including the C-fiber terminal, and might play a role in processing of visceral pain. In the present study, we examined and characterized the roles of PAR2 in pancreatitis-related abdominal hyperalgesia/allodynia in mice. 2 Caerulein, administered i.p. once, caused a small increase in abdominal sensitivity to stimulation with von Frey hairs, without causing pancreatitis, in PAR2-knockout (KO) mice, but not wild-type (WT) mice. 3 Caerulein, given hourly six times in total, caused more profound abdominal hyperalgesia/allodynia in PAR2-KO mice, as compared with WT mice, although no significant differences were detected in the severity of pancreatitis between the KO and WT animals. 4 The PAR2-activating peptide, 2-furoyl-LIGRL-NH(2), coadministered repeatedly with caerulein six times in total, abolished the caerulein-evoked abdominal hyperalgesia/allodynia in WT, but not PAR2-KO, mice. Repeated doses of 2-furoyl-LIGRL-NH(2) moderately attenuated the severity of caerulein-induced pancreatitis in WT animals. 5 Our data from experiments using PAR2-KO mice provide evidence that PAR2 functions to attenuate pancreatitis-related abdominal hyperalgesia/allodynia without affecting pancreatitis itself, although the PAR2AP applied exogenously is not only antinociceptive but also anti-inflammatory.

Abdominal Pain↗
PubMed2006

Granzyme K cleaves the nucleosome assembly protein SET to induce single-stranded DNA nicks of target cells.

Although granzymes (Gzms) A- and B-induced cell death pathways have been defined, little is known about how other orphan Gzms function in CTL-mediated cytotoxicity. GzmK and A are tryptases among all the Gzms of humans and they are closely linked on the same chromosome. In this study, we showed that GzmK can be efficiently delivered into target cells with a cationic lipid protein transfection reagent Pro-Ject. We found human GzmK triggers rapid cell death independently of caspase activation. The features of death are characterized by rapid externalization of phosphatidylserine, nuclear morphological changes and single-stranded DNA nicks. GzmK hydrolyzes the nucleosome assembly protein SET in its recombinant and native forms or in intact cells. Cleavage of SET by GzmK abrogates its nucleosome assembly activity. After GzmK loading, SET and DNase NM23H1 rapidly translocate into the nucleus and SET is cleaved, where the nuclease activity of NM23H1 is activated to nick chromosomal DNA.

Caspases↗
PubMed2001

Protease activated receptors: theme and variations.

The four PAR family members are G protein coupled receptors that are normally activated by proteolytic exposure of an occult tethered ligand. Three of the family members are thrombin receptors. The fourth (PAR2) is not activated by thrombin, but can be activated by other proteases, including trypsin, tryptase and Factor Xa. This review focuses on recent information about the manner in which signaling through these receptors is initiated and terminated, including evidence for inter- as well as intramolecular modes of activation, and continuing efforts to identify additional, biologically-relevant proteases that can activate PAR family members.

Amino Acid Sequence↗
PubMed2002

Myelomastocytic leukemia: myeloid neoplasm characterized by partial differentiation of mast cell-lineage cells.

A novel subtype of myeloid leukemia exhibiting a partial differentiation of mast cell-lineage cells is described. The disease is characterized by an increase in myeloblasts as well as an increase in immature (blast-like) metachromatic cells (>10% in bone marrow or blood smears). Metachromatic cells express KIT (CD117) and tryptase, but lack basophil-related antigens. In contrast to mast cell leukemia/systemic mastocytosis, metachromatic cells do not express CD2 or CD25, do not form multifocal dense aggregates in the bone marrow, and do not exhibit transforming mutations at codon 816 of c-kit. In the few patients recorded so far, a complex karyotype without recurring anomaly was found. The prognosis appears to be grave, although complete remission in response to chemotherapy has been described.

Cell Differentiation↗
PubMed2003

Purification and identification of a binding protein for pancreatic secretory trypsin inhibitor: a novel role of the inhibitor as an anti-granzyme A.

Pancreatic secretory trypsin inhibitor (PSTI) is a potent trypsin inhibitor that is mainly found in pancreatic juice. PSTI has been shown to bind specifically to a protein, distinct from trypsin, on the surface of dispersed cells obtained from tissues such as small intestine. In the present study, we affinity-purified the binding protein from the 2% (w/v) Triton X-100-soluble fraction of dispersed rat small-intestinal cells using recombinant rat PSTI. Partial N-terminal sequencing of the purified protein gave a sequence that was identical with the sequence of mouse granzyme A (GzmA), a tryptase produced in cytotoxic lymphocytes. We confirmed the formation of an affinity-cross-linked complex between (125)I-labelled PSTI and recombinant rat GzmA (rGzmA). In situ hybridization and immunostaining revealed the existence of GzmA-expressing intraepithelial lymphocytes in the rat small intestine. We concluded that the PSTI-binding protein isolated from the dispersed cells is GzmA that is produced in the lymphocytes of the tissue. The rGzmA hydrolysed the N -alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT), and the BLT hydrolysis was inhibited by PSTI. Sulphated glycosaminoglycans, such as fucoidan or heparin, showed almost no effect on the inhibition of rGzmA by PSTI, whereas they decreased the inhibition by antithrombin III. In the present paper, we propose a novel role of PSTI as a GzmA inhibitor.

Animals↗
PubMed2003

A novel serine protease predominately expressed in macrophages.

We have identified a novel serine protease designated EOS by sequence identity searches. The deduced protein contains 284 amino acids with an active form containing 248 amino acids starting from an Ile-Val-Gly-Gly motif. The active form comprises a catalytic triad of conserved amino acids: His77, Asp126 and Ser231. It shares 44% identity with beta-tryptase and belongs to the S1 trypsin-like serine-protease family. Interestingly, this gene also maps to human chromosome 16p13.3. The purified protease showed amidolytic activity, cleaving its substrates before arginine residues. Tissue distribution by immunohistochemistry analysis demonstrated that EOS is highly expressed in spleen and moderately expressed in intestine, colon, lung and brain. We confirmed this expression pattern at the mRNA level by performing in situ hybridization. The results from both immunohistochemistry and in situ hybridization indicate that EOS is associated with macrophages. We corroborated this observation by double immunofluorescence using the anti-EOS antibody and an anti-CD68 antibody, a macrophage specific marker. Furthermore, we have detected a dramatic increase in immune staining of EOS in cultured U937 cells treated with PMA, which represent activated macrophages. This up-regulation is also reflected by elevated EOS mRNA in the PMA-treated U937 cells detected by Northern blotting. Since macrophages have important roles in various pathological conditions, such as wound healing, atherosclerosis and numerous inflammatory diseases, the localization of this novel serine protease to active macrophages may help to further the elucidation of the roles of this gene product in modulating these disorders.

Amino Acid Sequence↗
PubMed2007

The crystal structure of human dipeptidyl peptidase I (cathepsin C) in complex with the inhibitor Gly-Phe-CHN2.

hDDPI (human dipeptidyl peptidase I) is a lysosomal cysteine protease involved in zymogen activation of granule-associated proteases, including granzymes A and B from cytotoxic T-lymphocytes and natural killer cells, cathepsin G and neutrophil elastase, and mast cell tryptase and chymase. In the present paper, we provide the first crystal structure of an hDPPI-inhibitor complex. The inhibitor Gly-Phe-CHN2 (Gly-Phe-diazomethane) was co-crystallized with hDPPI and the structure was determined at 2.0 A (1 A=0.1 nm) resolution. The structure of the native enzyme was also determined to 2.05 A resolution to resolve apparent discrepancies between the complex structure and the previously published structure of the native enzyme. The new structure of the native enzyme is, within the experimental error, identical with the structure of the enzyme-inhibitor complex presented here. The inhibitor interacts with three subunits of hDPPI, and is covalently bound to Cys234 at the active site. The interaction between the totally conserved Asp1 of hDPPI and the ammonium group of the inhibitor forms an essential interaction that mimics enzyme-substrate interactions. The structure of the inhibitor complex provides an explanation of the substrate specificity of hDPPI, and gives a background for the design of new inhibitors.

Cathepsin C↗
PubMed1983

Benzyl p-guanidinothiobenzoate hydrochloride, a new active-site titrant for trypsin and trypsin-like enzymes.

Benzyl p-guanidinothiobenzoate hydrochloride was synthesized and demonstrated to be useful for active-site titration of bovine trypsin, bovine thrombin, human lung tryptase, bovine activated protein C, human Factor XIIa fragment and bovine Factor Xa beta. The titration is based on rapid formation of a stable acyl-enzyme with a stoichiometric release of benzyl thiol. Thiol production is measured quantitatively by including 4,4'-dithiodipyridine in the reaction mixture and measuring the increase in absorbance at 324 nm. Ellman's reagent has also been successfully employed, allowing measurement at 410 nm. Unlike p-nitrophenyl p'-guanidinobenzoate, the thioester titrant reacts slowly with chymotrypsin A alpha thus eliminating interference by this enzyme in most titrations. Advantages of this reagent as a titrant include: flexibility in detection of the released thiol, selectivity between trypsin and chymotrypsin-like enzymes, minimal pH-dependence of the epsilon of the absorbing species, relative stability of the reagent under titration conditions, and high epsilon at pH 7.2 with either 4,4'-dithiodipyridine or Ellman's reagent. The reagent should prove useful as an alternative to p-nitrophenyl p'-guanidinobenzoate hydrochloride for the determination of active-site concentrations of the enzymes employed, as well as of other related enzymes.

Animals↗
PubMed1996

Cloning of the cDNAs for mast-cell chymases from the jejunum of Mongolian gerbils, Meriones unguiculatus, and their sequence similarities with chymases expressed in the connective-tissue mast cells of mice and rats.

By using the combination of reverse-transcription CR and rapid amplification of cDNA ends methods, two distinct cDNAs encoding mast-cell proteases (chymases; MCPs), designated as gMCP-1 and -2, were successfully cloned and sequenced from the jejunum of Mongolian gerbil, Meriones unguiculatus, infected with Nippostrongylus brasiliensis. On the basis of a comparison of the deduced amino acid sequences with those of known rodent mast-cell chymases, gMCP-1 was found to be highly similar to mouse mast-cell protease (mMCP)-4 and rat mast-cell protease (rMCP)-1, while gMCP-2 was similar to mMCP-5 and rMCP-3. Alghough mMCP-4 and -5 and rMCP-1 and -3 were restrictedly or mainly expressed in connective-tissue mast cells and serosal mast cells, the gMCP-1 and -2 genes were mainly transcribed in the jejunal mucosa and to a lesser extent in the skin and tongue. Moreover, kinetic study after infection revealed that the amounts of the gMCP-1 and -2 mRNAs in jejunum paralleled well the degree of intestinal mastocytosis. The expression of gMCP-1 and -2 in mucosal mast cells of gerbil jejunum was also confirmed by in situ hybridization. Since a tryptase, another type of MCP, was also expressed in mucosal mast cells of gerbils but not in those of mice and rats, the expression of MPCs in mucosal mast cells of gerbils is different from those of mice and rats. The Mongolian gerbil would be a useful model with which to investigate the physiopathological role of MCPs.

Amino Acid Sequence↗
PubMed2002

Protease-activated receptors: the role of cell-surface proteolysis in signalling.

Certain extracellular proteases, derived from the circulation and inflammatory cells, can specifically cleave and trigger protease-activated receptors (PARs), a small, but important, sub-group of the G-protein-coupled receptor super-family. Four PARs have been cloned and they all share the same basic mechanism of activation: proteases cleave at a specific site within the extracellular N-terminus to expose a new N-terminal tethered ligand domain, which binds to and thereby activates the cleaved receptor. Thrombin activates PAR1, PAR3 and PAR4, trypsin activates PAR2 and PAR4, and mast cell tryptase activates PAR2 in this manner. Activated PARs couple to signalling cascades that affect cell shape, secretion, integrin activation, metabolic responses, transcriptional responses and cell motility. PARs are 'single-use' receptors: proteolytic activation is irreversible and the cleaved receptors are degraded in lysosomes. Thus, PARs play important roles in 'emergency situations', such as trauma and inflammation. The availability of selective agonists and antagonists of protease inhibitors and of genetic models has generated evidence to suggests that proteases and their receptors play important roles in coagulation, inflammation, pain, healing and protection. Therefore, selective antagonists or agonists of these receptors may be useful therapeutic agents for the treatment of human diseases.

Animals↗
PubMed2003

Protease-activated receptor 2: activation, signalling and function.

PARs (protease-activated receptors) are a family of four G-protein-coupled receptors for proteases from the circulation, inflammatory cells and epithelial tissues. This report focuses on PAR(2), which plays an important role in inflammation and pain. Pancreatic (trypsin I and II) and extrapancreatic (trypsin IV) trypsins, mast cell tryptase and coagulation factors VIIa and Xa cleave and activate PAR(2). Proteases cleave PAR(2) to expose a tethered ligand that binds to the cleaved receptor. Despite this irreversible activation, PAR(2) signalling is attenuated by beta-arrestin-mediated desensitization and endocytosis, and by lysosomal targeting and degradation, which requires ubiquitination of PAR(2). beta-Arrestins also act as scaffolds for the assembly of multi-protein signalling complexes that determine the location and function of activated mitogen-activated protein kinases. Observations of PAR(2)-deficient mice support a role for PAR(2) in inflammation, and many of the effects of PAR(2) activators promote inflammation. Inflammation is mediated in part by activation of PAR(2) in the peripheral nervous system, which results in neurogenic inflammation and hyperalgesia.

Animals↗
PubMed2002

Agonists of proteinase-activated receptor 2 induce cytokine release and activation of nuclear transcription factor kappaB in human dermal microvascular endothelial cells.

Proteinase-activated receptor 2 belongs to a new G protein-coupled receptor subfamily activated by various serine proteases. It has been demonstrated to play a role during inflammation of many tissues including the skin. Proteinase-activated receptor 2 is expressed by endothelial cells and regulates cutaneous inflammation in vivo. The underlying mechanisms of proteinase-activated receptor 2 activation in the skin and the effects on human dermal microvascular endothelial cells, however, are still unknown. Agonists of proteinase-activated receptor 2 such as mast cell tryptase induce widespread inflammation in many organs including the skin. Trypsinogen is generated by endothelial cells during inflammation or tumor growth. Therefore we tested whether human dermal microvascular endothelial cells express functional proteinase-activated receptor 2 and whether agonists of proteinase-activated receptor 2 regulate inflammatory responses in these cells. Calcium mobilization studies revealed that proteinase-activated receptor 2 is functional in human dermal microvascular endothelial cells. Interleukin-6 and interleukin-8 were upregulated as detected by reverse transcription polymerase chain reaction or enzyme-linked immunosorbent assay indicating a role of proteinase-activated receptor 2 in stimulating human dermal microvascular endothelial cells. Electromobility shift assays revealed proteinase-activated-receptor-2-induced activation of nuclear transcription factor kappaB with a maximum after 1 h. In conclusion, agonists of proteinase-activated receptor 2 upregulate interleukin-6 and interleukin-8 expression and release in human dermal microvascular endothelial cells. Thus, proteinase-activated receptor 2 may play an important role in cutaneous inflammation by mediating inflammatory responses on dermal microvascular endothelial cells and activation of nuclear transcription factor kappaB.

Cells, Cultured↗
PubMed2000

Angiogenesis in wound healing.

During wound healing, angiogenic capillary sprouts invade the fibrin/fibronectin-rich wound clot and within a few days organize into a microvascular network throughout the granulation tissue. As collagen accumulates in the granulation tissue to produce scar, the density of blood vessels diminishes. A dynamic interaction occurs among endothelial cells, angiogenic cytokines, such as FGF, VEGF, TGF-beta, angiopoietin, and mast cell tryptase, and the extracellular matrix (ECM) environment. Specific endothelial cell ECM receptors are critical for these morphogenetic changes in blood vessels during wound repair. In particular, alpha(v)beta3, the integrin receptor for fibrin and fibronectin, appears to be required for wound angiogenesis: alpha(v)beta3 is expressed on the tips of angiogenic capillary sprouts invading the wound clot, and functional inhibitors of alpha(v)beta3 transiently inhibit granulation tissue formation. Recent investigations have shown that the wound ECM can regulate angiogenesis in part by modulating integrin receptor expression. mRNA levels of alpha(v)beta3 in human dermal microvascular endothelial cells either plated on fibronectin or overlaid by fibrin gel were higher than in cells plated on collagen or overlaid by collagen gel. Wound angiogenesis also appears to be regulated by endothelial cell interaction with the specific three-dimensional ECM environment in the wound space. In an in vitro model of human sprout angiogenesis, three-dimensional fibrin gel, simulating early wound clot, but not collagen gel, simulating late granulation tissue, supported capillary sprout formation. Understanding the molecular mechanisms that regulate wound angiogenesis, particularly how ECM modulates ECM receptor and angiogenic factor requirements, may provide new approaches for treating chronic wounds.

Animals↗