Mast cell activation in response to osmotic and immunological stimulation with focus on release of eicosanoid mediators

Abstract: Mast cells are important in asthma and other inflammatory diseases. Subjects with asthma have been found to have an increased number of mast cells in their airway smooth muscle and this was related to airway sensitivity. Normally harmless stimuli may trigger bronchoconstriction in subjects with asthma and exercise can generate airway constriction in subjects with asthma. The mechanism for exercise-induced bronchoconstriction (EIB) has been suggested to be related to an increased airway fluid osmolarity. This may activate mast cells with subsequent release of mediators acting on bronchial smooth muscle leading to bronchoconstriction. Mannitol inhalation causes bronchoconstriction, and the mechanism is probably by increasing airway fluid osmolarity. The aim of this thesis was to establish whether hyperosmolar stimulation activates human mast cells in vitro and in vivo with focus on the release of biologically active mediators. Human cord blood derived mast cells (CBMC) were used for studies on mediator release in response to immunological and osmotic activation in vitro. Bronchial provocation by mannitol inhalation was used to mimic EIB for studies in vivo on airway reactivity and urinary excretion of mediators. For the first time, mannitol was found to induce the release of PGD2 and LTC4 in CBMC in vitro. Prostaglandin D2 was formed both via the COX-1 and COX-2 pathways in CBMC. The late response after stimulation with the combination of anti- IgE and IL-1beta was more COX-2 dependent. Further, the pro-inflammatory cytokine IL-1beta induced the expression of COX-2. In addition to COX derived PGD2, CBMC was found to release TXB2 and occasionally also PGE2 after stimulation with IL-1beta, anti-IgE or their combination. Hypoxia (4% O2) was not found to increase the release of mediators as compared to normoxic (21% O2) conditions. Interleukin-4 induced the expression of 15-LO in CBMC and the main 15-LO derived metabolite was 15-KETE followed by 15-HETE in IL-4 treated CBMC stimulated with arachidonic acid. The release of 15-HETE was also induced by mannitol Both asthmatic and control subjects had an increased urinary excretion of the PGD2 metabolite 9alpha,11beta-PGF2 as well as LTE4 after mannitol challenge in vivo. The increase in 9alpha,11beta-PGF2 was related to bronchoconstriction since only the asthmatic subjects responded to mannitol. Further, the mast cell stabiliser sodium cromoglycate (SCG) and the beta2-agonist formoterol protected from mannitol-inducedbronchoconstriction in asthmatic subjects with 63% and 95%, respectively. In addition, both inhibitors dampened the mannitol-induced urinary 9alpha,11beta-PGF2 excretion compared to placebo treatment. In conclusion, mast cells release PGD2 after mannitol stimulation in vitro and in vivo and treatment with a mast cell stabiliser further supports the mast cell involvement in mannitol-induced bronchoconstriction in vivo. Both COX-1 and COX-2 enzymes were involved in PGD2 formation and mast cells were unaffected by hypoxic environmental changes in vitro. The expression of 15-LO in mast cells in vivo and in vitro support that these cells can contribute to the formation of novel metabolites with unknown functions. The mediator formation in mast cells seems to be important for subjects with EIB since their airways respond more easily with bronchoconstriction. Inhibition of PGD2 formation protects from bronchoconstriction in subjects with EIB. The physiological effect of some mast cell mediators remains to be elucidated however PGD2 appear to have a central role in the airway response to mannitol.