Azelastine inhibits secretion of IL-6, TNF-alpha and IL-8 as well as NF-kappaB activation and intracellular calcium ion levels in normal human mast cells.

BACKGROUND: Mast cells are involved in allergic inflammation by secreting histamine, proteases and several cytokines, including interleukin (IL)-6, tumor necrosis factor-alpha (TNF-alpha) and IL-8. Certain histamine-1 receptor antagonists, such as azelastine present in the ophthalmic solution Optivar, have been reported to inhibit histamine and tryptase secretion, but its effect on inflammatory cytokine release from normal human umbilical cord blood-derived cultured mast cells (hCBMC) are not well known. METHODS: We investigated the effect of azelastine on the secretion of IL-6, TNF-alpha and IL-8 from hCBMC, as well as its possible mechanism of action. hCBMC sensitized with IgE were pretreated for 5 min with azelastine at 0.01, 0.1, 1, 3, 6, 12, 24, or 60 microM of Optivar, before stimulation with anti-IgE for 6 h. Optivar vehicle without azelastine was used as control. Cytokines were measured by ELISA, intracellular calcium levels by calcium indicators confocal, and nuclear factor-kappaB (NF-kappaB) by electromobility shift assay. RESULTS: Stimulation with anti-IgE led to substantial secretion of IL-6, TNF-alpha and IL-8. Preincubation for 5 min resulted in almost maximal inhibition with 6 microM azelastine for TNF-alpha (80%), with 24 microM for IL-6 (83%) and 60 microM for IL-8 (99%); the vehicle solution at the same concentrations as Optivar had no effect. Stimulation with anti-IgE increased intracellular Ca2+ level and induced NF-kappaB expression in hCBMC, which was inhibited by 24 microM azelastine. CONCLUSION: Azelastine inhibited hCBMC secretion of IL-6, TNF-alpha and IL-8, possibly by inhibiting intracellular Ca2+ ions and NF-kappaB activation. Azelastine may, therefore, be helpful in treating allergic inflammation.

Corticotropin-releasing factor (CRF) can directly affect brain microvessel endothelial cells.

Stress activates the hypothalamic-pituitary-adrenal (HPA) axis through release of corticotropin releasing factor (CRF), leading to production of glucocorticoids that down regulate immune responses. However, acute stress via CRF also has pro-inflammatory effects. We previously showed that acute stress increases rat blood-brain barrier (BBB) permeability, an effect involving brain mast cells and CRF, as it was absent in W/W(v) mast cell-deficient mice and was blocked by the CRF-receptor antagonist, Antalarmin. We investigated if CRF could also have a direct action on brain microvessel endothelial cells (BMEC) isolated from rat and bovine brain. BMEC were cultured and identified by electron microscopy. Western blot analysis of cultured BMEC identified CRF receptor protein; stimulation with CRF, or it structural analogue urocortin (Ucn) showed that the receptor is functionally coupled to adenylate cyclase as it increased cyclic AMP (cAMP) levels by 2-fold. These findings suggest that CRF could affect BMEC structure or function, as reported for increased cAMP levels by other studies. It is, therefore, possible that CRF may directly regulate BBB permeability, in addition to any effect mediated via brain mast cells.

Corticotropin-releasing hormone and brain mast cells regulate blood-brain-barrier permeability induced by acute stress.

Stress activates the hypothalamic-pituitary-adrenal axis through release of corticotropin releasing hormone (CRH), leading to production of glucocorticoids that down-regulate immune responses. Acute stress, however, also has proinflammatory effects that seem to be mediated through the activation of mast cells. Stress and mast cells have been implicated in the pathophysiology of various inflammatory conditions, including some in the central nervous system, such as multiple sclerosis in which disruption of the blood-brain barrier (BBB) precedes clinical symptoms. We previously showed that acute restraint stress increases rat BBB permeability to intravenous 99Tc gluceptate and that administration of the "mast cell stabilizer" disodium cromoglycate (cromolyn) inhibits this effect. In this study, we show that the CRH-receptor antagonist Antalarmin blocks stress-induced 99Tc extravasation, whereas site-specific injection of CRH in the paraventricular nucleus (PVN) of the hypothalamus mimics acute stress. This latter effect is blocked by pretreatment of the PVN with cromolyn; moreover, restraint stress cannot disrupt the BBB in the diencephalon and cerebellum of W/W(v) mast cell-deficient mice. These results demonstrate that CRH and mast cells are involved in regulating BBB permeability and, possibly, brain inflammatory disorders exacerbated by acute stress.