Mast cells (MCs) are important in allergic diseases, tissue injury, and protection from infections. Indirect evidence suggests that the effector function of MCs is controlled by T cells, yet little is understood concerning this process in humans due to the limitations of human studies in vivo, and to technical difficulties in the isolation, purification, and maintenance in culture. Cysteinyl leukotrienes (cys-LTs), a class of peptide- conjugated lipids generated by MCs, signal through at least two known 7 transmembrane-spanning, G protein-coupled receptors (GPCR), termed the CysLT1 and CysLT2 receptors. Each is homologous to the purinergic (P2Y) receptors for nucleotides. Human MCs (hMCs) express the CysLT1 receptor, which unexpectedly mediates activation responses to both cys-LTs and to the extracellular nucleotide agonist uridine diphosphate (UDP). Furthermore, priming of hMCs with recombinant IL-4 preferentially enhances their sensitivity to both LTC4 and UDP without a concomitant change in CysLT1 receptor expression, suggesting that either a third cys-LT receptor (CysLT3) is induced by IL-4, or that the fundamental pharmacologic properties of CysLT1 are altered by IL-4 priming so as to selectively lower its threshold for LTC4 and UDP binding over LTD4. We cys-LTs and UDP induce the generation of multiple cytokines (EL-5, macrophage inflammatory protein 1beta, and tumor necrosis factor [TNF]- alpha) by IL-4-primed hMCs, and that blockade of endogenous cys- LT receptors or interference with endogenous cys-LT synthesis attenuate the generation of both IL-5 and TNF-alpha in response to cross-linking of the high-affinity Fc receptor for IgE (Fc epsilon RI). We propose the following hypotheses: 1). Cys-LTs mediate both autocrine and paracrine functions of hMCs through at least two GPCRs that also bind uridine nucleotides, linking neurogenic signals and tissue injury to MCs and inflammation in asthma; 2) these same receptors permit MCs to initiate inflammatory responses to injury and infection in other tissues.
Specific Aim 1 focuses on identifying the CysLT3 receptor and defining the mechanism(s) responsible for the observed IL-4- induced upregulation of hMC responses to UDP and LTC4.
Specific Aim 2 uses site-directed mutagenesis to define the regions of the CysLT1 (and possible CysLT3) receptors involved in binding of LTD4, LTC4, and UDP, respectively.
Specific Aim 3 will explore the functional role of the GPCRs for cys-LT and UDP in mediating MC-dependent inflammatory responses in vivo using targeted deletion of their respective genes.
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