thapsigargin and Anaphylaxis

Mast cells play a pivotal role in IgE-mediated allergic responses. Development of specific inhibitors against FcεRI-associated proximal signaling molecules in mast cells may represent a promising therapeutic strategy for allergic diseases. We examined whether a novel synthetic compound, 3-butyl-1-chloro-8-(2-methoxycarbonyl)phenyl-5H-imidazo[1,5-b]isoquinolin-10-one (U63A05), could suppress antigen-stimulated degranulation and cytokine secretion in mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. U63A05 reversibly and dose-dependently inhibited degranulation of rat basophilic leukemia (RBL)-2H3 mast cells and bone marrow-derived mast cells (BMMCs) stimulated by antigen (IC(50) values for RBL-2H3 and BMMCs were 4.1 and 4.8 µM, respectively). The secretion of inflammatory cytokines was also suppressed in antigen-stimulated mast cells. However, degranulation by thapsigargin, a typical calcium inducer, was not inhibited by U63A05. U63A05 exerts its inhibitory effect, to the same extent as in degranulation, on the activating phosphorylation of Syk and downstream signaling molecules, including LAT and SLP-76. Further downstream, the activating phosphorylations of Akt, Erk1/2, p38, and JNK were also inhibited. Finally, antigen-stimulated PCA was dose-dependently suppressed in mice (ED(50), 26.3 mg/kg). Taken together, the results suggest that U63A05 suppresses the activation of mast cells and the mast cell-mediated allergic response through the inhibition of Syk activation in mast cells.

Topics: Anaphylaxis; Animals; Cell Degranulation; Cell Line; Cytokines; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Drug Interactions; Imidazoles; Immunoglobulin E; Intracellular Signaling Peptides and Proteins; Isoquinolines; Male; Mast Cells; Mice; Mice, Inbred BALB C; Passive Cutaneous Anaphylaxis; Phosphorylation; Protein-Tyrosine Kinases; Rats; Signal Transduction; Syk Kinase; Thapsigargin

Inflammatory responses by mast cells are characterized by massive exocytosis of prestored granular mediators followed by cytokine/chemokine release. The vesicular trafficking mechanisms involved remain poorly understood. Vesicular-associated membrane protein-8 (VAMP-8), a member of the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) family of fusion proteins initially characterized in endosomal and endosomal-lysosomal fusion, may also function in regulated exocytosis. Here we show that in bone marrow-derived mast cells (BMMCs) VAMP-8 partially colocalized with secretory granules and redistributed upon stimulation. This was associated with increased SNARE complex formation with the target t-SNAREs, SNAP-23 and syntaxin-4. VAMP-8-deficient BMMCs exhibited a markedly reduced degranulation response after IgE+ antigen-, thapsigargin-, or ionomycin-induced stimulation. VAMP-8-deficient mice also showed reduced plasma histamine levels in passive systemic anaphylaxis experiments, while cytokine/chemokine release was not affected. Unprocessed TNF accumulated at the plasma membrane where it colocalized with a VAMP-3-positive vesicular compartment but not with VAMP-8. The findings demonstrate that VAMP-8 segregates secretory lysosomal granule exocytosis in mast cells from cytokine/chemokine molecular trafficking pathways.

Topics: Anaphylaxis; Animals; Antigens; Cell Degranulation; Cytokines; Exocytosis; Histamine; Immunoglobulin E; Inflammation; Ionomycin; Ionophores; Lactones; Lysosomes; Mast Cells; Membrane Fusion; Mice; Mice, Knockout; Protein Transport; Qa-SNARE Proteins; Qb-SNARE Proteins; R-SNARE Proteins; Secretory Vesicles; Thapsigargin

The anti-allergic action of various Oriental medicinal herbs was investigated using in vitro and in vivo experimental models. Of these extracts, the ethanol extract of Meliae cortex (MC) exhibited the most potent activity in mast cells; its IC(50) values were 29+/-1.5 microg/ml for antigen stimulation and 57+/-3.4 microg/ml for thapsigargin stimulation. It inhibited compound-48/80-induced systemic anaphylaxis by 52.9% at a dose of 300 mg/kg in mice; it also inhibited the expression of the proinflammatory mediator TNF-alpha. With regard to its mechanism of action, MC suppressed the activating phosphorylation of Syk, a key enzyme in mast-cell signaling processes and that of Akt in a dose-dependent manner. It also inhibited the MAP kinase ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of the activating phosphorylation of ERK1/2. Taken together, these results suggest that the anti-allergic activity of MC may be due to the inhibition of histamine secretion and cytokine expression through the Syk inhibition in mast cells.

Topics: Anaphylaxis; Animals; Anti-Allergic Agents; Cell Degranulation; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Gene Expression; Immunoblotting; Intracellular Signaling Peptides and Proteins; Male; Mast Cells; Melia azedarach; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Mitogen-Activated Protein Kinases; p-Methoxy-N-methylphenethylamine; Phosphorylation; Plant Extracts; Protein-Tyrosine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Syk Kinase; Thapsigargin; Tumor Necrosis Factor-alpha