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Intercellular Ca²⁺ wave propagation involving positive feedback between CRAC channels and cysteinyl leukotrienes

Joseph Di Capite^*, Anna Shirley^*, Charmaine Nelson^*, Grant Bates and Anant B. Parekh^*,1

^* Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and

Department of Ear, Nose and Throat Surgery, John Radcliffe Hospital, Oxford, UK

¹Correspondence: Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford OX1 3PT, UK. E-mail: anant.parekh{at}dpag.ox.ac.uk

Mast cells are key components of the immune system, where they help orchestrate the inflammatory response. Aberrant mast cell activation is linked to a variety of allergic diseases, including asthma, eczema, rhinitis, and nasal polyposis, which in combination affect up to 20% of the population in industrialized countries. On activation, mast cells release a variety of signals that target the bronchi and vasculature and recruit other immune cells to the inflammatory site. Prominent among such signals are the cysteinyl leukotrienes, a family of potent proinflammatory lipid mediators comprising leukotriene C₄ (LTC₄), LTD₄, and LTE₄. LTC₄, the parent compound, is secreted from mast cells following Ca²⁺ influx through store-operated calcium release-activated calcium (CRAC) channels. Here, we show that activated mast cells release a paracrine signal that evokes Ca²⁺ signals in spatially separate resting mast cells. The paracrine signal was identified as a cysteinyl leukotriene because 1) RNAi knockdown or pharmacological block of the 5-lipoxygenase enzyme prevented activated mast cells from stimulating resting cells. 2) Block of cysteinyl leukotriene type I receptors on resting mast cells with the clinically prescribed receptor antagonist montelukast prevented their activation by active mast cells. 3) RNAi knockdown of cysteinyl leukotriene type I receptors on resting cells prevented them from responding to the paracrine signal derived from activated mast cells. 4) Purified LTC₄ evoked Ca²⁺ signals in mast cells that were identical to those triggered by the paracrine signal. Low levels of stimulus intensity released sufficient levels of leukotriene to activate resting cells. Leukotriene secretion still occurred tens of minutes after stimulation, suggesting a role as a long-lasting trigger in mast cell activation. Stimulation of the cysteinyl leukotriene receptor activated CRAC channels and evoked prominent store-operated Ca²⁺ entry. This resulted in further cysteinyl leukotriene production, triggering a positive feedback cascade. Acutely isolated mast cells from patients with allergic rhinitis exhibited store-operated Ca²⁺ influx through CRAC channels and responded to cysteinyl leukotrienes. Histological analysis of samples taken from patients revealed clustering of mast cells, often located within 20 µm of each other, a distance sufficient for paracrine signaling by leukotrienes to operate effectively. We conclude that a positive-feedback cascade involving CRAC channels and cysteinyl leukotrienes constitute a novel mechanism for sustaining mast cell activation.—Di Capite, J., Shirley, A., Nelson, C., Bates, G., Parekh, A. B. Intercellular Ca²⁺ wave propagation involving positive feedback between CRAC channels and cysteinyl leukotrienes.

Key Words: paracrine signal • mast cells