IL-4 amplifies the pro-inflammatory effect of adenosine in human mast cells by changing expression levels of adenosine receptors

Adenosine inhalation produces immediate bronchoconstriction in asthmatics but not in normal subjects. The bronchospastic effect of adenosine is largely mediated through adenosine-induced mast cell activation, the mechanism of which is poorly understood due to limitations in culturing human primary mast cells. Here, we show that human umbilical cord blood -derived mast cells incubated with the Th2 cytokine IL-4 develop increased sensitivity to adenosine. Potentiation of anti-IgE- induced and calcium ionophore/PMA-induced degranulation was augmented in mast cells cultured with IL-4, and this effect was reduced or abolished by pre-treatment with A(2B)siRNA and selective A(2B) receptor antagonists, respectively. IL-4 incubation resulted in the increased expression of A(2B) and reduced expression of A(2A) adenosine receptors on human mast cells. These results suggest that Th2 cytokines in the asthmatic lung may alter adenosine receptor expression on airway mast cells to promote increased responsiveness to adenosine.     

Functional transient receptor potential melastatin 7 channels are critical for human mast cell survival

Mast cells play a significant role in the pathophysiology of many diverse diseases such as asthma and pulmonary fibrosis. Ca2+ influx is essential for mast cell degranulation and release of proinflammatory mediators, while Mg2+ plays an important role in cellular homeostasis. The channels supporting divalent cation influx in human mast cells have not been identified, but candidate channels include the transient receptor potential melastatin (TRPM) family. In this study, we have investigated TRPM7 expression and function in primary human lung mast cells (HLMCs) and in the human mast cell lines LAD2 and HMC-1, using RT-PCR, patch clamp electrophysiology, and RNA interference. Whole cell voltage-clamp recordings revealed a nonselective cation current that activated spontaneously following loss of intracellular Mg2+. The current had a nonlinear current-voltage relationship with the characteristic steep outward rectification associated with TRPM7 channels. Reducing external divalent concentration from 3 to 0.3 mM dramatically increased the size of the outward current, whereas the current was markedly inhibited by elevated intracellular Mg2+ (6 mM). Ion substitution experiments revealed cation selectivity and Ca2+ permeability. RT-PCR confirmed the presence of mRNA for TRPM7 in HLMC, LAD2, and HMC-1 cells. Adenoviral-mediated knockdown of TRPM7 in HLMC with short hairpin RNA and in HMC-1 with short interfering RNA markedly reduced TRPM7 currents and induced cell death, an effect that was not rescued by raising extracellular Mg2+. In summary, HLMC and human mast cell lines express the nonselective cation channel TRPM7 whose presence is essential for cell survival.     

Adenosine-activated mast cells induce IgE synthesis by B lymphocytes: an A2B-mediated process involving Th2 cytokines IL-4 and IL-13 with implications for asthma

Adenosine provokes bronchoconstriction in asthmatics through acute activation of mast cells, but its potential role in chronic inflammation has not been adequately characterized. We hypothesized that adenosine up-regulates Th2 cytokines in mast cells, thus promoting IgE synthesis by B lymphocytes. We tested this hypothesis in human mast cells (HMC-1) expressing A(2A), A(2B), and A(3) adenosine receptors. The adenosine analog 5'-N-ethylcarboxamidoadenosine (NECA) (10 microM) increased mRNA expression of IL-1beta, IL-3, IL-4, IL-8, and IL-13, but not IL-2 and IFN-gamma. Up-regulation of IL-4 and IL-13 was verified using RT-PCR and ELISA; 10 microM NECA increased IL-13 concentrations in HMC-1 conditioned medium 28-fold, from 7.6 +/- 0.3 to 215 +/- 4 pg/ml, and increased IL-4 concentrations 6-fold, from 19.2 +/- 0.1 to 117 +/- 2 pg/ml. This effect was mediated by A(2B) receptors because neither the selective A(2A) agonist 2-p-(2-carboxyethyl)phenethylamino-NECA nor the selective A(3) agonist N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine reproduced it, and the selective A(2B) antagonist 3-isobutyl-8-pyrrolidinoxanthine prevented it. Constitutive expression of CD40 ligand on HMC-1 surface was not altered by NECA. Human B lymphocytes cocultured for 12 days with NECA-stimulated HMC-1 produced 870 +/- 33 pg IgE per 10(6) B cells, whereas lymphocytes cocultured with nonstimulated HMC-1, or cultured alone in the absence or in the presence of NECA, produced no IgE. Thus, we demonstrated induction of IgE synthesis by the interaction between adenosine-stimulated mast cells and B lymphocytes, and suggest that this mechanism is involved in the amplification of the allergic inflammatory responses associated with asthma.     

Evidence for a novel Kit adhesion domain mediating human mast cell adhesion to structural airway cells

Background

Human lung mast cells (HLMCs) infiltrate the airway epithelium and airway smooth muscle (ASM) in asthmatic airways. The mechanism of HLMC adhesion to both cell types is only partly defined, and adhesion is not inhibited by function-blocking anti-Kit and anti-stem cell factor (SCF) antibodies. Our aim was to identify adhesion molecules expressed by human mast cells that mediate adhesion to human ASM cells (HASMCs) and human airway epithelial cells.

Methods

We used phage-display to isolate single chain Fv (scFv) antibodies with adhesion-blocking properties from rabbits immunised with HLMC and HMC-1 membrane proteins.

Results

Post-immune rabbit serum labelled HLMCs in flow cytometry and inhibited their adhesion to human BEAS-2B epithelial cells. Mast cell-specific scFvs were identified which labelled mast cells but not Jurkat cells by flow cytometry. Of these, one scFv (A1) consistently inhibited mast cell adhesion to HASMCs and BEAS-2B epithelial cells by about 30 %. A1 immunoprecipitated Kit (CD117) from HMC-1 lysates and bound to a human Kit-expressing mouse mast cell line, but did not interfere with SCF-dependent Kit signalling.

Conclusion

Kit contributes to human mast cell adhesion to human airway epithelial cells and HASMCs, but may utilise a previously unidentified adhesion domain that lies outside the SCF binding site. Targeting this adhesion pathway might offer a novel approach for the inhibition of mast cell interactions with structural airway cells, without detrimental effects on Kit signalling in other tissues.     

Antibody-directed fibrinolysis. An antibody specific for both fibrin and tissue plasminogen activator

An investigation was made to determine whether it is possible to attract tissue plasminogen activator (tPA) to the site of a thrombus by means of an antibody with affinites for both tPA and fibrin. A bispecific antibody conjugate was constructed by cross-linking two monoclonal antibodies: one specific for tPA, the other specific for fibrin. The bispecific antibody enhanced fibrinolysis by capturing tPA at the site of a fibrin deposit. In an in vitro quantitative fibrinolysis assay, the relative fibrinolytic potency of tPA bound to the bispecific antibody was 13 times greater than that of tPA and 200 times greater than that of urokinase. When fibrin was treated with the bispecific antibody before being mixed with tPA, the relative fibrinolytic potency of tPA was enhanced 14-fold. This capture also occurred when the concentration of tPA present in the assay was less than the concentration of tPA present in normal human plasma. In a human plasma clot assay, samples containing both the bispecific antibody and tPA exhibited significantly more lysis than did samples containing tPA alone. In spite of the increased clot lysis effected by the presence of bispecific antibody, there was no significant increase in fibrinogen or alpha 2-antiplasmin degradation at equal tPA concentrations. The ability of the bispecific antibody to concentrate exogenous tPA in vivo was then examined in the rabbit jugular vein model. Systemic infusion of a small amount of tPA (10,000 units) produced no significant increment in thrombolysis over the level of spontaneous lysis (14 +/- 8%). However, the simultaneous infusion of 10,000 units of tPA and 2 mg of bispecific antibody resulted in 42 +/- 14% (p less than 0.01) lysis. These results suggest that a molecule capable of binding both fibrin and tPA with high affinity could enhance thrombolysis in the circulation by capturing endogenous tPA.     

Thymic stromal lymphopoietin is regulated by the intracellular calcium

The cytokine thymic stromal lymphopoietin (TSLP) has been implicated in the development and progression of allergic diseases such as atopic dermatitis. However, it has not been clarified that TSLP would be regulated by intracellular calcium in mast cells yet. To determine it, we blocked intracellular calcium by treatment with calcium chelator, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) in human mast cell line (HMC-1) cells. BAPTA-AM inhibited the production and mRNA expression of TSLP in phorbol myristate acetate plus A23187- stimulated HMC-1 cells. BAPTA-AM also inhibited the nuclear factor-κB activation, IκBα phosphorylation, receptor interacting protein2 (RIP2) expression, and caspase-1 activation in HMC-1 cells. These results provide evidence that calcium regulates the level of TSLP through RIP2/caspase-1/NF-κB/IκBα signal.     

CADM1 Is a Key Receptor Mediating Human Mast Cell Adhesion to Human Lung Fibroblasts and Airway Smooth Muscle Cells

Background

Mast cells (MCs) play a central role in the development of many diseases including asthma and pulmonary fibrosis. Interactions of human lung mast cells (HLMCs) with human airway smooth muscle cells (HASMCs) are partially dependent on adhesion mediated by cell adhesion molecule-1 (CADM1), but the adhesion mechanism through which HLMCs interact with human lung fibroblasts (HLFs) is not known. CADM1 is expressed as several isoforms (SP4, SP1, SP6) in HLMCs, with SP4 dominant. These isoforms differentially regulate HLMC homotypic adhesion and survival.

Objective

In this study we have investigated the role of CADM1 isoforms in the adhesion of HLMCs and HMC-1 cells to primary HASMCs and HLFs.

Methods

CADM1 overexpression or downregulation was achieved using adenoviral delivery of CADM1 short hairpin RNAs or isoform-specific cDNAs respectively.

Results

Downregulation of CADM1 attenuated both HLMC and HMC-1 adhesion to both primary HASMCs and HLFs. Overexpression of either SP1 or SP4 isoforms did not alter MC adhesion to HASMCs, whereas overexpression of SP4, but not SP1, significantly increased both HMC-1 cell and HLMC adhesion to HLFs. The expression level of CADM1 SP4 strongly predicted the extent of MC adhesion; linear regression indicated that CADM1 accounts for up to 67% and 32% of adhesion to HLFs for HMC-1 cells and HLMCs, respectively. HLFs supported HLMC proliferation and survival through a CADM1-dependent mechanism. With respect to CADM1 counter-receptor expression, HLFs expressed both CADM1 and nectin-3, whereas HASMCs expressed only nectin-3.

Conclusion and Clinical Relevance

Collectively these data indicate that the CADM1 SP4 isoform is a key receptor mediating human MC adhesion to HASMCs and HLFs. The differential expression of CADM1 counter-receptors on HLFs compared to HASMCs may allow the specific targeting of either HLMC-HLF or HLMC-HASMC interactions in the lung parenchyma and airways.     

Methallyl isothiocyanate inhibits the caspase-1 activity through the inhibition of intracellular calcium levels

Isothiocyanates (ITCs) play a major role in the potential chemopreventive effect. Cruciferous vegetables are a particularly abundant source of ITCs. Methallyl ITC (MAITC) belongs to ITCs as a synthesized compound. However, the effects of MATIC have never been elucidated. The aim of this work was to investigate the anti-inflammatory effects and mechanisms of methallyl isothiocyanate (MAITC) in mast cells. MAITC suppressed the intracellular calcium levels in phorbol myristate acetate (PMA) plus calcium ionophore A23187 (PMACI)-stimulated human mast cell line (HMC-1) cells. MAITC significantly inhibited the production and mRNA expression of interleukin (IL)-1β in PMACI-stimulated HMC-1 cells. The activities of caspase-1 and receptor interacting protein-2 were significantly inhibited by the treatment with MAITC in PMACI-stimulated HMC-1 cells. The activation of nuclear factor-κB and phosphorylation of extracellular signal-regulated kinase and IκBα were inhibited by the treatment with MAITC. In addition, MAITC significantly inhibited the production and mRNA expression of IL-6 and tumor necrosis factor-α in PMACI-stimulated HMC-1 cells. Furthermore, MAITC significantly inhibited caspase-1 enzymatic activity and reactive oxygen species generation in PMA-stimulated HMC-1 cells. Taken together, we can conclude that MAITC showed an anti-inflammatory effect on mast cell-mediated inflammatory reaction.     

Effect of A2B adenosine receptor gene ablation on proinflammatory adenosine signaling in mast cells

Pharmacological studies suggest that A(2B) adenosine receptors mediate proinflammatory effects of adenosine in human mast cells in part by up-regulating production of Th2 cytokines and angiogenic factors. This concept has been recently challenged by the finding that mast cells cultured from bone marrow-derived mast cells (BMMCs) of A(2B) knockout mice display an enhanced degranulation in response to FcepsilonRI stimulation. This finding was interpreted as evidence of anti-inflammatory functions of A(2B) receptors and it was suggested that antagonists with inverse agonist activity could promote activation of mast cells. In this report, we demonstrate that genetic ablation of the A(2B) receptor protein has two distinct effects on BMMCs, one is the previously reported enhancement of Ag-induced degranulation, which is unrelated to adenosine signaling; the other is the loss of adenosine signaling via this receptor subtype that up-regulates IL-13 and vascular endothelial growth factor secretion. Genetic ablation of A(2B) receptors had no effect on A(3) adenosine receptor-dependent potentiation of Ag-induced degranulation in mouse BMMCs, but abrogated A(2B) adenosine receptor-dependent stimulation of IL-13 and vascular endothelial growth factor secretion. Adenosine receptor antagonists MRS1706 and DPCPX with known inverse agonist activity at the A(2B) subtype inhibited IL-13 secretion induced by the adenosine analog NECA, but did not mimic the enhanced Ag-induced degranulation observed in A(2B) knockout BMMCs. Thus, our study confirmed the proinflammatory role of adenosine signaling via A(2B) receptors and the anti-inflammatory actions of A(2B) antagonists in mouse BMMCs.     

High resolution analysis of functional determinants on human tissue-type plasminogen activator

Sixty-four variants of human tissue-type plasminogen activator (tPA) were produced using recombinant DNA techniques. Charged residues were converted to alanine in clusters of from one to four changes per variant; these clusters spanned all the domains of the molecule. The variants were expressed by mammalian cells and were analyzed for a variety of properties. Variants of tPA were found that had reduced activity with respect to each tested property; in a few cases increased activity was observed. Analysis of these effects prompted the following conclusions: 1) charged residues in the nonprotease domains are less involved in fibrin stimulation of tPA activity than those in the protease domain, and it is possible to increase the fibrin specificity (i.e. the stimulation of tPA activity by fibrin compared to fibrinogen) by mutations at several sites in the protease domain; 2) the difference in enzymatic activity between the one- and two-chain forms of tPA can be increased by mutations at several sites on the protease domain; 3) binding of tPA to lysine-Sepharose was affected only by mutations to kringle-2, whereas binding to fibrin was affected most by mutations in the other domains; 4) clot lysis was influenced by mutations in all domains except kringle-2; 5) sensitivity to plasminogen activator inhibitor-1 seems to reside exclusively in the region surrounding residue 300. A model of the tPA protease domain has been used to map some of the critical residues and regions.     

Activation of murine lung mast cells by the adenosine A3 receptor

Adenosine has been implicated to play a role in asthma in part through its ability to influence mediator release from mast cells. Most physiological roles of adenosine are mediated through adenosine receptors; however, the mechanisms by which adenosine influences mediator release from lung mast cells are not understood. We established primary murine lung mast cell cultures and used real-time RT-PCR and immunofluorescence to demonstrate that the A(2A), A(2B), and A(3) adenosine receptors are expressed on murine lung mast cells. Studies using selective adenosine receptor agonists and antagonists suggested that activation of A(3) receptors could induce mast cell histamine release in association with increases in intracellular Ca(2+) that were mediated through G(i) and phosphoinositide 3-kinase signaling pathways. The function of A(3) receptors in vivo was tested by exposing mice to the A(3) receptor agonist, IB-MECA. Nebulized IB-MECA directly induced lung mast cell degranulation in wild-type mice while having no effect in A(3) receptor knockout mice. Furthermore, studies using adenosine deaminase knockout mice suggested that elevated endogenous adenosine induced lung mast cell degranulation by engaging A(3) receptors. These results demonstrate that the A(3) adenosine receptor plays an important role in adenosine-mediated murine lung mast cell degranulation.     

Conjugation to an antifibrin monoclonal antibody enhances the fibrinolytic potency of tissue plasminogen activator in vitro

Tissue plasminogen activator (tPA) was covalently linked by disulfide bonds to a monoclonal antibody specific for the amino terminus of the beta chain of fibrin (antibody 59D8). The activity of the tPA-59D8 conjugate was compared with that of tPA, urokinase (UK), and a UK-59D8 conjugate. For lysis of fibrin monomer, tPA was 10 times as potent as UK, whereas both UK-59D8 and tPA-59D8 conjugates were 100 times as potent as UK and 10 times as potent as tPA. Conjugation of tPA or UK to antibody 59D8 produced a 3.2-4.5-fold enhancement in clot lysis in human plasma over that of the respective unconjugated plasminogen activator. However, the UK-59D8 conjugate was only as potent as tPA alone. Antibody-conjugated tPA or UK consumed less fibrinogen, alpha 2-antiplasmin, and plasminogen than did the unconjugated activators, at equipotent fibrinolytic concentrations. Antibody targeting thus appears to increase the concentration of tPA in the vicinity of a fibrin deposit, which thereby leads to enhanced fibrinolysis.     

Tryptase activates the mitogen-activated protein kinase/activator protein-1 pathway in human peripheral blood eosinophils,causing cytokine production and release

We have previously shown that mast cells enhance eosinophil survival and activation. In this study we further characterized mast cell activity toward eosinophils. Sonicate of both rat peritoneal mast cells and the human mast cell line 1 (HMC-1) induced a concentration-dependent IL-6 and IL-8 release from human peripheral blood eosinophils (ELISA). HMC-1-induced IL-8 release was significantly reduced by the tryptase inhibitors GW-45 and GW-58 (90 and 87%, respectively, at an optimal concentration) but not by anti-stem cell factor, anti-TNF-alpha, or anti-IFN-gamma neutralizing Abs or by the antihistamine drugs pyrilamine and cimetidine. In a manner similar to HMC-1, human recombinant tryptase induced the expression of mRNA for IL-8 (RT-PCR) and caused IL-8 release from the eosinophils. Addition of cycloheximide, actinomycin D, dexamethasone, PD 98059, curcumin, or SB 202190 completely inhibited the tryptase-induced IL-6 and IL-8 release. In contrast, cyclosporin A had no effect on tryptase-induced IL-8 release. Tryptase caused phosphorylation of extracellular signal-regulated kinases 1 and 2, c-Jun N-terminal kinases 1 and 2, and p38 (Western blot). Tryptase also induced the translocation of c-Jun from the cytosol to the nucleus (confocal microscopy) and enhanced AP-1 binding activity to the DNA (EMSA). Eosinophils were found to express proteinase-activated receptor 2 (FACS). When eosinophils were incubated with tryptase in the presence of anti-proteinase-activated receptor 2 antagonist Abs a significant decrease in the IL-6 and IL-8 release occurred. In summary, we have demonstrated that the preformed mast cell mediator tryptase induces cytokine production and release in human peripheral blood eosinophils by the mitogen-activated protein kinase/AP-1 pathway.     

A3 adenosine receptor activation inhibits cell proliferation via phosphatidylinositol 3-kinase/Akt-dependent inhibition of the extracellular signal-regulated kinase 1/2 phosphorylation in A375 human melanoma cells

Adenosine exerts its effects through four subtypes of G-protein-coupled receptors: A(1), A(2A), A(2B), and A(3). Stimulation of the human A(3) receptor has been suggested to influence cell death and proliferation. The phosphatidylinositide-3-OH kinase (PI3K)/Akt and the Raf/mitogen-activated protein kinase (MAPK/ERK) kinase (MEK)/mitogen-activated protein kinase (MAPK) pathways have central roles in the regulation of cell survival and proliferation. Due to their importance, the cross-talk between these two pathways has been investigated. Here, we show that the A(3) adenosine receptor agonist Cl-IB-MECA stimulates PI3K-dependent phosphorylation of Akt leading to the reduction of basal levels of ERK1/2 phosphorylation, which in turn inhibits cell proliferation. The response to Cl-IB-MECA was not blocked by A(1), A(2A), or A(2B) receptor antagonists, although it was abolished by A(3) receptor antagonists. Furthermore, the response to Cl-IB-MECA was generated at the cell surface, since the inhibition of A(3) receptor expression, by using small interfering RNA, abolished agonist effects. Using A375 cells, we show that A(3) adenosine receptor stimulation results in PI3K-dependent phosphorylation of Akt, leading to the reduction of basal levels of ERK1/2 phosphorylation, which in turn inhibits cell proliferation.     

IL-4 regulates c-kit proto-oncogene product expression in human mast and myeloid progenitor cells.

The c-kit proto-oncogene encodes the receptor for a novel hemopoietic cytokine, termed stem cell factor (SCF) or mast cell growth factor (MGF) according to its stimulating spectrum. The human receptor for SCF/MGF is expressed in a subset of normal bone marrow progenitor cells, in leukemic myeloid cells, and in mast cells. In the present study, the effects of recombinant human growth regulators (IL-1 through -9, granulocyte-macrophage/granulocyte/macrophage-CSF, IFN, and TNF) on c-kit proto-oncogene product expression were analyzed by indirect immunofluorescence, by using the anti-SCF/MGFR mAb YB5.B8, and Northern blot analyses, by using a c-kit oligonucleotide probe. Of all cytokines tested, IL-4 was found to down-regulate expression of YB5.B8 Ag in the human mast cell line HMC-1 (maximum inhibition, 51.05 +/- 16.36% mean fluorescence intensity of control; p less than 0.02), as well as in primary leukemic myeloid cells. IL-4 was also found to down-regulate expression of YB5.B8 Ag in normal enriched bone marrow progenitor cells. The effects of IL-4 on expression of YB8.B8 Ag in myeloid/mast cell progenitors was dose and time dependent (maximum effects observed on days 2 and/or 4, by using 50 U/ml of rIL-4) and could be neutralized by using anti-IL-4 mAb. Moreover, IL-4 was found to down-regulate expression of c-kit mRNA in leukemic myeloid cells as well as in HMC-1 cells. Together, these observations identify IL-4 as a regulator of c-kit proto-oncogene product expression in the human system. The effects of IL-4 on human hemopoietic progenitor cells and mast cells may be mediated in part through regulation of SCF/MGFR expression.     

Primary human airway epithelial cell-dependent inhibition of human lung mast cell degranulation

Introduction

Chronic mast cell activation is a characteristic feature of asthma. BEAS-2B human airway epithelial cells (AEC) profoundly inhibit both constitutive and IgE-dependent human lung mast cell (HLMC) histamine release. The aim of this study was to examine the regulation of HLMC degranulation by primary AEC from healthy and asthmatic subjects, and investigate further the inhibitory mechanism.

Methods

HLMC were co-cultured with both BEAS-2B and primary AEC grown as monolayers or air-liquid interface (ALI) cultures.

Results

Both constitutive and IgE-dependent HLMC histamine release were attenuated by BEAS-2B, primary AEC monolayers and ALI cultures. This occurred in the absence of HLMC-AEC contact indicating the presence of a soluble factor. Unlike healthy ALI AEC, asthmatic ALI-AEC did not significantly reduce constitutive histamine release. AEC inhibitory activity was transferable in primary AEC monolayer supernatant, but less active than with Transwell co-culture, suggesting that the inhibitory factor was labile. The AEC inhibitory effects were attenuated by both AEC wounding and pertussis toxin, indicating the involvement of a G₀/G_i receptor coupled mechanism. Solid phase extraction of lipids (<10 kDa) removed the AEC inhibitory activity. The lipid derivatives resolvin D1 and D2 and lipoxin A₄ attenuated HLMC histamine release in a dose-dependent fashion but were not detectable in co-culture supernatants.

Conclusions

Primary AEC suppress HLMC constitutive and IgE-dependent histamine secretion through the release of a soluble, labile lipid mediator(s) that signals through the G₀/G_i receptor coupled mechanism. Manipulation of this interaction may have a significant therapeutic role in asthma.     

Tissue-type plasminogen activator is a multiligand cross-beta structure receptor

Tissue-type plasminogen activator (tPA) regulates fibrin clot lysis by stimulating the conversion of plasminogen into the active protease plasmin. Fibrin is required for efficient tPA-mediated plasmin generation and thereby stimulates its own proteolysis. Several fibrin regions can bind to tPA, but the structural basis for this interaction is unknown. Amyloid beta (Abeta) is a peptide aggregate that is associated with neurotoxicity in brains afflicted with Alzheimer's disease. Like fibrin, it stimulates tPA-mediated plasmin formation. Intermolecular stacking of peptide backbones in beta sheet conformation underlies cross-beta structure in amyloid peptides. We show here that fibrin-derived peptides adopt cross-beta structure and form amyloid fibers. This correlates with tPA binding and stimulation of tPA-mediated plasminogen activation. Prototype amyloid peptides, including Abeta and islet amyloid polypeptide (IAPP) (associated with pancreatic beta cell toxicity in type II diabetes), have no sequence similarity to the fibrin peptides but also bind to tPA and can substitute for fibrin in plasminogen activation by tPA. Moreover, the induction of cross-beta structure in an otherwise globular protein (endostatin) endows it with tPA-activating potential. Our results classify tPA as a multiligand receptor and show that cross-beta structure is the common denominator in tPA binding ligands.     

Human airway smooth muscle promotes human lung mast cell survival, proliferation, and constitutive activation: cooperative roles for CADM1, stem cell factor, and IL-6

The microlocalization of mast cells within specific tissue compartments is thought to be critical for the pathophysiology of many diverse diseases. This is particularly evident in asthma where they localize to the airway smooth muscle (ASM) bundles. Mast cells are recruited to the ASM by numerous chemoattractants and adhere through CADM1, but the functional consequences of this are unknown. In this study, we show that human ASM maintains human lung mast cell (HLMC) survival in vitro and induces rapid HLMC proliferation. This required cell-cell contact and occurred through a cooperative interaction between membrane-bound stem cell factor (SCF) expressed on ASM, soluble IL-6, and CADM1 expressed on HLMC. There was a physical interaction in HLMC between CADM1 and the SCF receptor (CD117), suggesting that CADM1-dependent adhesion facilitates the interaction of membrane-bound SCF with its receptor. HLMC-ASM coculture also enhanced constitutive HLMC degranulation, revealing a novel smooth muscle-driven allergen-independent mechanism of chronic mast cell activation. Targeting these interactions in asthma might offer a new strategy for the treatment of this common disease.     

Investigation into mechanisms mediating the inhibitory effect of 1,4-benzodiazepines on mast cells by gene expression profiling

AimsThis study aims to identify by a molecular genetic approach potential targets in mast cells at which 1,4-benzodiazepines may cause their inhibitory effect on mast cell activity.Main methodsGene expression analyses with microarray gene chip and/or quantitative PCR were performed using 1,4-benzodiazepine-treated human mast cell leukemia HMC-1.2 cells, promyelocytic leukemia HL-60 cells and human mast cells from healthy volunteers and patients with mast cell activation disease (MCAD). Pathway analysis was applied to search for enriched biological functions and canonical pathways within differentially regulated genes.Key findingsBoth neoplastic and normal human mast cells express several GABA_A receptor subunits at the mRNA level. In mast cells from MCAD patients expression of some GABA_A receptor subunits and expression of the translocator protein TSPO are increased compared with those from healthy controls. Expression of the protein tyrosine kinases Lyn, Fgr and Yes1 was increased in HMC-1.2 cells as compared with the ontogenetically related HL60 cells. Differences in gene regulation in HMC-1.2 cells after treatment with the 1,4-benzodiazepines clonazepam, flunitrazepam and 4-chlorodiazepam suggested that signaling and gene expression induced by clonazepam was similar to that of flunitrazepam but different from that of 4-chlorodiazepam. This conclusion is supported by the results of the pathway analysis.SignificanceA novel type of GABA_A receptors on mast cells appears to be involved in the inhibition of mast cell activity by 1,4-benzodiazepines. These receptors seem to be composed without γ subunits suggesting unique pharmacological properties. An action at Src-kinases, or at TSPO located in the plasma membrane may also be involved.