Mucosal mast cells (MMCs) have an important role in allergic inflammation, and effective antagonists are required for their regulation. To discover a possible mechanism of controlling the activation of MMCs, we investigated the expression and function of syntaxin4, one of the soluble membrane N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, in RBL-2H3 cells, which is a rat mucosal mast cell line. Syntaxin4 silencing was induced by transfection of short interfering RNAs (siRNAs). Syntaxin4 was knocked down in mast cells at both the mRNA and protein levels. The release of granule contents that are involved in inflammation, such as histamine and hexosaminidase, was significantly suppressed by the gene silencing of syntaxin4. Silencing of this gene was also induced in the trachea and bronchi of rats by intratracheal application of the siRNAs using an atelocollagen delivery system. The activation of MMCs, which was monitored by the level of rat mast cell protease-II (RMCPII) in the bronchoalveolar lavage fluid (BALF), was inhibited, and asthmatic airway constriction was prevented by administration of the syntaxin/atelocollagen complex. These results indicate that siRNAs targeting syntaxin4 can stabilize mucosal mast cells and may have beneficial therapeutic effects on the asthmatic response. To identify a possible mechanism for controlling the activation of MMCs, we focused on the mast cell exocytotic step within the complex intracellular signal transduction pathway of MMC activation. Exocytosis is the process by which signal transduction between plasma membrane receptors and vesicular membrane proteins results in the release of inflammatory mediators from cytoplasmic granules into the extracellular environment. The fusion of secretory granules with the plasma membrane is the final step in a series of biochemical events that leads to the release of inflammatory mediators from mast cells.Mast cells are equipped with specific signalling molecules, the functions of which contribute to the fusion of vesicles with the plasma membrane bilayer. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) comprise a large family of membrane fusion proteins. 4 The interaction between SNARE proteins on the vesicle membrane (v-SNARE) and those on the target membrane (t-SNARE) is considered to promote membrane fusion in mast cells. In MMCs, four types of t-SNAREs (syntaxin2, syntaxin3 and syntaxin4, and SNAP23) and four types of v-SNAREs (vesicle-associated membrane protein-2 (VAMP2), 3, 7 and 8) can be detected at the protein level. 5 In our pilot study, of the three kinds of syntaxins, only syntaxin3 and syntaxin4 showed changes in expression in stimulated MMCs. The complex of syntaxin3 and VAMPs has already been well described in previous studies. 6 However, the regulation of syntaxin3 did not completely abolish the exocytosis of MMCs. We are trying to find new targets to regulate the function of MMCs.We investigated the function of syntaxin4, one of the t-SNAREs, in inflammat...
The regulated control of Ca(2+) influx is essential for the activation and function of the adaptive immune response, as Ca(2+) is a key regulator of important transcription factors. To determine whether Ca(2+) release-activated Ca(2+) (CRAC) channels contribute to the abnormal behaviour of T cells in patients with rheumatoid arthritis (RA), we performed a cross-sectional study to characterize the expression and functional status of CRACM1 channels in RA patients. Peripheral blood was obtained from 50 RA patients, 50 osteoarthritis (OA) patients and healthy donors. We measured Ca(2+) influx and CRAC currents in naïve and memory CD4(+) T cells. CRACM1 expression was evaluated in T cells from each of the three groups. These cells were further characterized by flow cytometric analysis of interleukin-4 (IL-4), IL-17, interferon-γ and tumour necrosis factor-α. These cytokines were also measured in naïve CD4(+) T cells following the lentivirus-mediated silencing of CRACM1.There was a significant positive correlation between Ca(2+) influx in naïve T cells and RA activity. Functionally aberrant naïve CD4(+) T cells from patients with active RA showed the different cytokine release pattern and exhibited increased Ca(2+) influx as well as increased CRACM1 protein expression and function. Specific lentiviral-induced gene silencing of CRACM1 reversed the alterations in T-cell cytokine production. The data presented here indicate that an upregulation of CRACM1 expression and function may be responsible for the abnormal cytokine release of naïve CD4(+) T cells in RA patients. CRACM1 might therefore represent a new molecular target for RA therapies.
Store-operated Ca release-activated Ca (CRAC) channels are involved in the pathogenesis of rheumatoid arthritis (RA) and have been studied as therapeutic targets in the management of RA. We investigated the efficacy and safety of CRAC inhibitors, including a neutralizing Ab (hCRACM1-IgG) and YM-58483, in the treatment of RA. Patient-derived T cell and B cell activity was suppressed by hCRACM1-IgG as well as YM-58483. Systemically constant, s.c. infused CRAC inhibitors showed anti-inflammatory activity in a human-NOD/SCID xenograft RA model as well as protective effects against the destruction of cartilage and bone. hCRACM1-IgG appeared to be safe for systemic application, whereas YM-58483 showed hepatic and renal toxicity in xenograft mice. Treatment with both CRAC inhibitors also caused hyperglycemia in xenograft mice. These results indicate the potential of hCRACM1-IgG and YM-58483 as anti-immunological agents for the treatment of RA. However, some safety issues should be addressed and application methods should be optimized prior to their clinical use.
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