| Mast cells are crucial effector cells in host defense and immunoregulation. They are equipped with an impressive array of receptor systems which allows them to rapidly respond to pathogen-associated signals. G protein-coupled receptors (GPCRs) mediate mast cell activation and mediator release in response to various stimuli. The effects are differential; while some GPCR can induce chemotaxis, adhesion, degranulation and mediator release, others can stimulate chemotaxis and adhesion but not degranulation. Signaling through GPCRs can also modulate antigen-mediated activation of mast cells. The effect mediated via a GPCR depends on the G protein it couples with. Signal transduction via Galphai initiates cell activation, whereas Galphas downregulates cell activation. My thesis investigated the roles of novel stimulatory and inhibitory GPCRs in human mast cells. I hypothesized that human mast cells express both stimulatory and inhibitory chemoattractant GPCRs which differentially activate mast cells but chemotaxis pathway itself remains conserved. For the first sub-hypothesis, expression and function of FPRL1 receptor in human mast cells was investigated. I showed that Laboratory of Allergic Diseases 2 (LAD2) human mast cells expressed cell surface FPRL1. Stimulation of LAD2 cells with a novel alpha-cationic antimicrobial peptide, pleurocidin, induced mast cell chemotaxis, adhesion, degranulation and production of lipid mediators, cytokines, and chemokines. The effect was sensitive to pertussis toxin, indicating that FPRL1 mediates stimulatory signaling in human mast cells via Galpha i protein.;In the next sub-hypothesis, expression and function of C5a receptors, C5aR and C5L2, in human mast cells was investigated. While C5aR is a known Galphai-coupled GPCR in human mast cells, the signaling mechanism for C5L2 is unknown. My data showed that LAD2 human mast cells express cell surface C5L2 but not C5aR. C5a activated LAD2 cells to migrate, adhere and produce cytokines and chemokines. The effects were abolished in mast cells in which C5L2 expression was knocked-down using lentiviral C5L2 shRNA. Moreover, the effect of C5a on cell adhesion was pertussis toxin-dependent, indicating that C5L2 mediates stimulatory signaling in human mast cells via Ga, proteins.;In the final sub-hypothesis, function of C3a receptor, C3aR, in human mast cells was investigated in conjugation with adenosine receptor signaling. Stimulatory signal transduction through Galphai coupled C3aR in human mast cells is well-established. To test the expression and function of inhibitory GPCRs, adenosine receptors were utilized. I showed that while adenosine did not activate human mast cells by itself, it inhibited C3amediated activation of human mast cell adhesion, chemotaxis, degranulation, and chemokine production. An A2A receptor specific agonist, CGS 21680, blocked the inhibitory effect of adenosine, indicating that Galphas-coupled adenosine receptor exert inhibitory effects on Galphai-coupled GPCR activation in human mast cells.;Overall, my thesis research provides crucial information on the effects of novel chemoattractant GPCRs in human mast cells. While these receptors differentially modulated various human mast cell functions either positively via Galphas-coupled GPCRs or negatively via Galphai-coupled GPCRs, chemotaxis remained conserved and occurred via a Gag dependent signal transduction mechanism. |
