Abstract
Association of the neurotransmitter serotonin (5-HT) with the pathogenesis of allergic asthma is well recognized and its role as a chemoattractant for eosinophils (Eos) in vitro and in vivo has been previously demonstrated. Here we have examined the regulation of 5-HT-induced human and murine Eos trafficking and migration at a cellular and molecular level. Eos from allergic donors and bone marrow-derived murine Eos (BM-Eos) were found to predominantly express the 5-HT2A receptor. Exposure to 5-HT or 2,5-dimethoxy-4-iodoamphetamine (DOI), a 5-HT2A/C selective agonist, induced rolling of human Eos and AML14.3D10 human Eos-like cells on vascular cell adhesion molecule (VCAM)-1 under conditions of flow in vitro coupled with distinct cytoskeletal and cell shape changes as well as phosphorylation of MAPK. Blockade of 5-HT2A or of ROCK MAPK, PI3K, PKC and calmodulin, but not Gαi-proteins, with specific inhibitors inhibited DOI-induced rolling, actin polymerization and changes in morphology of VCAM-1-adherent AML14.3D10 cells. More extensive studies with murine BM-Eos demonstrated the role of 5-HT in promoting rolling in vivo within inflamed post-capillary venules of the mouse cremaster microcirculation and confirmed that down-stream signaling of 5-HT2A activation involves ROCK, MAPK, PI3K, PKC and calmodulin similar to AML14.3D10 cells. DOI-induced migration of BM-Eos is also dependent on these signaling molecules and requires Ca2+. Further, activation of 5-HT2A with DOI led to an increase in intracellular Ca2+ levels in murine BM-Eos. Overall, these data demonstrate that 5-HT (or DOI)/5-HT2A interaction regulates Eos trafficking and migration by promoting actin polymerization associated with changes in cell shape/morphology that favor cellular trafficking and recruitment via activation of specific intracellular signaling molecules (ROCK, MAPK, PI3K and the PKC-calmodulin pathway).
Highlights
DOI-induced Eos migration is dependent on ROCK, MAPK, PI3K, PKC and calmodulin Since 5-HT is a known chemoattractant for human Eos and allergen-challenged mice treated with cyproheptadine (a 5-HT2A inhibitor) exhibit decreased Eos recruitment [11], we examined whether 5-HT and DOI induce murine Eos migration and how this is regulated
Studies have clearly established a role for this molecule in promoting allergen-induced Eos recruitment, airway inflammation, airway hyperresponsiveness (AHR) and remodeling [11,24,25], the hallmarks of allergic asthma, via interaction with the 5-HT2A receptor
Current RT-PCR studies indicate that 5-HT2A is the predominant 5-HT receptor expressed by human Eos from allergic donors, the level of expression of this receptor is variable among different donors
Summary
5-Hydroxytryptamine (5-HT, serotonin) is one of the most extensively studied neurotransmitters of the central nervous system (CNS) which is known to have a multitude of physiological functions outside the CNS These include stimulation of cytokine [1,2,3,4] and chemokine production [4,5], vasoconstriction [6], tissue regeneration [7], cell (fibroblasts, smooth muscle cells [SMC], endothelial cells) proliferation [8,9,10] and migration (eosinophils [Eos], mast cells [MC], SMC, dendritic cells [DC]) [4,11,12,13], and regulation of the immune system [14]. We have used a systematic approach enabling interpretation of cross-species findings between human and murine Eos to examine how 5-HT regulates various aspects of Eos trafficking (rolling and adhesion in vitro and in vivo within inflamed blood vessels) and migration including the role of specific signaling molecules involved in these events
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