Background Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid produced by mast cells (MC) upon cross-linking of their high affinity receptors for IgE by antigen (Ag) that can amplify MC responses by binding to its S1P receptors. Acute MC-dependent allergic reaction can lead to systemic shock but the early events of its development in lung tissues have not been investigated, and S1P functions in the onset of allergic processes remain to be examined. Objective We used a highly specific neutralizing anti-S1P antibody (mAb) and an S1P receptor 2 (S1PR2) antagonist, JTE-013, to study S1P and S1PR2 signaling contributions to MC- and IgE-dependent airway allergic responses in mice within minutes after Ag challenge. Methods Allergic reaction was triggered by a single intraperitoneal (i.p.) dose of Ag in sensitized mice pre-treated i.p. with anti-S1P or isotype control mAb, or JTE-013 or vehicle prior to Ag challenge. Results Kinetics experiments revealed early pulmonary infiltration of mostly T cells around blood vessels of sensitized mice 20 minutes post-Ag exposure. Pre-treatment with anti-S1P mAb inhibited in vitro MC activation, as well as in vivo development of airway infiltration and MC activation, reducing serum levels of histamine, cytokines and the chemokines MCP-1/CCL2, MIP-1α/CCL3 and RANTES/CCL5. S1PR2 antagonism or deficiency, or MC deficiency recapitulated these results. Both in vitro and in vivo experiments demonstrated MC S1PR2 dependency for chemokine release and the necessity for signal transducer and activator of transcription 3 (Stat3) activation. Conclusion Activation of S1PR2 by S1P and downstream Stat3 signaling in MC regulate early T cell recruitment to antigen-challenged lungs by chemokine production.

We describe MC and S1P as novel pathogenic effectors that initiate remodeling in AD prior to any skin lesions and reveal the significance of LPS in OVA used in most studies, thus mimicking natural antigen (Ag) exposure.

Apart from their effector functions in allergic disorders, tissue-resident mast cells (MC) are gaining recognition as initiators of inflammatory events through their distinctive ability to secrete many bioactive molecules harbored in cytoplasmic granules. Activation triggers mediator release through a regulated exocytosis named degranulation. MC activation is still substantiated by measuring systemic levels of MC-restricted mediators. However, identifying the anatomical location of MC activation is valuable for disease diagnosis. We designed a computer-assisted morphometric method based on image analysis of methylene blue (MB)-stained normal mouse skin tissue sections that quantitates actual in situ MC activation status. We reasoned MC cytoplasm could be viewed as an object featuring unique relative mass values based on activation status. Integrated optical density and area (A) ratios were significantly different between intact and degranulated MC (p<0.001). The examination of fractal characteristics is of translational diagnostic/prognostic value in cancer and readily applied to quantify cytoskeleton morphology and vasculature. Fractal dimension (D), a measure of their comparative space filling capacity and structural density, also differed significantly between intact and degranulated MC (p<0.001). Morphometric analysis provides a reliable and reproducible method for in situ quantification of MC activation status.

Mast cells (MC) are present in most vascularized tissues around the vasculature likely exerting immunomodulatory functions. Endowed with diverse mediators, resident MC represent first-line fine-tuners of local microenvironment. Sphingosine-1-phosphate (S1P) functions as a pluripotent signaling sphingolipid metabolite in health and disease. S1P formation occurs at low levels in resting MC and is upregulated upon activation. Its export can result in type 2 S1P receptor- (S1PR2-) mediated stimulation of MC, further fueling inflammation. However, the role of S1PR2 ligation in proangiogenic vascular endothelial growth factor- (VEGF-) A and matrix metalloproteinase- (MMP-) 2 release from MC is unknown. Using a preclinical MC-dependent model of acute allergic responses and in vitro stimulated primary mouse bone marrow-derived MC (BMMC) or human primary skin MC, we report that S1P signaling resulted in substantial amount of VEGF-A release. Similar experiments using S1pr2-deficient mice or BMMC or selective S1P receptor agonists or antagonists demonstrated that S1P/S1PR2 ligation on MC is important for VEGF-A secretion. Further, we show that S1P stimulation triggered transcriptional upregulation of VEGF-A and MMP-2 mRNA in human but not in mouse MC. S1P exposure also triggered MMP-2 secretion from human MC. These studies identify a novel proangiogenic axis encompassing MC/S1P/S1PR2 likely relevant to inflammation.

Mast cells produce a potently bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) constitutively and upon activation. The ligation of S1P to its type 2 receptor on mast cells triggers a novel downstream signaling pathway that we discovered links activation of transcription factor signal transducer and activator of transcription 3 to mast cell-derived chemokine release in both humans and mice. In this chapter, we describe the methods used to study S1P signaling in human and mouse primary mast cells.