Mast Cell-mediated Inflammatory Reactions Research Articles

Blockade of RANKL/RANK signaling pathway by epigallocatechin gallate alleviates mast cell-mediated inflammatory reactions

Receptor activator of NF-κB ligand (RANKL) as an osteoclast differentiation factor induces inflammatory reactions via production of thymic stromal lymphopoietin (TSLP). Epigallocatechin gallate (EGCG) is the major and the most active compound in green tea and has anti-inflammatory, anti-cancer, anti-oxidant, and neuroprotective effects. However, the effect and molecular mechanisms of EGCG are still unknown in RANKL-induced inflammatory reactions. Here we investigated the immuno-regulatory effects and its molecular mechanisms of epigallocatechin gallate (EGCG) in RANKL-stimulated human mast cell line, HMC-1 cells. In this study, EGCG prevented expression of PI3 Kinase and phosphorylation of mitogen-activated protein (MAP) Kinases in RANKL-stimulated HMC-1 cells. EGCG prevented caspase-1 activity and decreased transcriptional activity of nuclear factor (NF)-κB by suppressing inhibitory protein κBα phosphorylation in RANKL-stimulated HMC-1 cells. EGCG has been shown to prevent production and mRNA expression of TSLP, interleukin (IL)-1β, IL-6, and IL-8 by RANKL without cytotoxicity. Furthermore, EGCG prevented degranulation of mast cell in RANKL-stimulated HMC-1 cells. Overall, these results suggest that EGCG acts as a natural agent for preventing and treating RANKL-mediated inflammatory diseases by targeting PI3 Kinase, MAP Kinase, caspase-1, and NF-κB signaling cascade in mast cells.

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.

Involvement of MITF-A, an alternative isoform ofmitranscription factor, on the expression of tryptase gene in human mast cells

Mast cells play a central role in the initiation and development of allergic diseases through release of various mediators. Tryptase has been known to be a key mediator in mast cell-mediated inflammatory reactions. In the present study, we investigated whether the transcription of tryptase gene in human mast cells was induced by microphthalmia (mi)-associated transcription factor (MITF). We observed that the human CD34+ progenitor-derived cultured mast cells and human mast cell line HMC-1 expressed strongly the transcripts of tryptase-beta1 and MITF-A, which is a MITF alterative splicing isoform. The transcriptional activity of tryptase gene was specifically higher in HMC-1 cells compared to the tryptase-negative cells. Using mutant constructs of tryptase promoter, we observed that two E-box (CANNTG) motifs including between -817 to -715 and -421 to -202 are able to involve in the transactivation of tryptase gene by MITF-A. In addition, the binding of these motifs-containing oligonucleotides to MITF proteins was detectable by EMGA using the nuclear extracts of HMC-1 cells and anti-MITF mAb. The overexpression of MITF-A elevated tryptase production by HMC-1 cells, while the introduction of specific siRNA against MITF attenuated the expression and enzymatic activity of tryptase. These data suggest that MITF might play a role in regulating the transcription of tryptase gene in human mast cells.

Requirement of c-jun transcription factor on the mouse mast cell protease-6 expression in the mast cells

Mast cell tryptases may be a key mediator in mast cell-mediated inflammatory reactions, and these expressions can be regulated by microenvironmental factors of tissues, particularly stem cell factor. In the present study, we investigated whether the transcription of mouse mast cell protease-6 (mMCP-6) gene was caused by SCF-mediated c-jun. We observed that mMCP-6 mRNA was expressed by overexpression of c-jun in the immature mast cell line in which both mMCP-6 and c-kit receptor are negative. The c-jun increased synergistically the luciferase activity of mMCP-6 promoter through the direct interaction with mi transcription factor (MITF). The synergic effect of c-jun with MITF was abolished by deletion of sequence between nt −171 and −151 in the mMCP-6 promoter. Furthermore, the level of mMCP-6 mRNA in mast cells was attenuated by the introduction of dominant negative c-jun (TAM-67) and the treatment of Jun N-terminal kinase inhibitor, SP600125. These results show that c-jun might play a role in regulating the transcription of mMCP-6 gene in mast cells stimulated by SCF.

Heparin antagonists are potent inhibitors of mast cell tryptase.

Tryptase may be a key mediator in mast cell-mediated inflammatory reactions. When mast cells are activated, they release large amounts of these tetrameric trypsin-like serine proteases. Tryptase is present in a macromolecular complex with heparin proteoglycan where the interaction with heparin is known to be essential for maintaining enzymatic activity. Recent investigations have shown that tryptase has potent proinflammatory activity, and inhibitors of tryptase have been shown to modulate allergic reactions in vivo. Many of the tryptase inhibitors investigated previously are directed against the active site. In the present study we have investigated an alternative approach for tryptase regulation. We show that the heparin antagonists Polybrene and protamine are potent inhibitors of both human lung tryptase and of recombinant mouse tryptase (mouse mast cell protease 6). Protamine inhibited tryptase in a competitive manner whereas Polybrene showed noncompetitive inhibition kinetics. Treatment of tetrameric, active tryptase with Polybrene caused dissociation into monomers, accompanied by complete loss of enzymatic activity. The present report thus suggests that heparin antagonists potentially may be used in treatment of mast cell-mediated diseases such as asthma.

The Tryptase, Mouse Mast Cell Protease 7, Exhibits Anticoagulant Activity in Vivo and in Vitro Due to Its Ability to Degrade Fibrinogen in the Presence of the Diverse Array of Protease Inhibitors in Plasma

Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, we used this in vivo model system to identify a physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with immunoglobulin (Ig) E and challenged with antigen were found to contain substantial amounts of four 34-55-kDa peptides, all of which were derived from fibrinogen. To confirm the substrate specificity of mMCP-7, a pseudozymogen form of the recombinant tryptase was generated that could be activated after its purification. The resulting recombinant mMCP-7 exhibited potent anticoagulant activity in the presence of normal plasma and selectively cleaved the alpha-chain of fibrinogen to fragments of similar size as that seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptase-specific, phage display peptide library revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the alpha-chain of rat fibrinogen. Because fibrinogen is a physiologic substrate of mMCP-7, this tryptase can regulate clot formation and fibrinogen/integrin-dependent cellular responses during mast cell-mediated inflammatory reactions.