Studies on the role of IL-33-induced 5-lipoxygenase-dependent signals and Fc epsilon R1-interacting IQGAP1 in mast cells

Mast cells (MCs) are sentinel cells of the immune system, residing in locations of interface between the body and environment. MCs function as initiators of defense against pathogens, however, they are also well known for their roles in allergic diseases. MCs are equipped with a host of receptors, including the high-affinity receptor for IgE, Fc epsilon R1, along with members of the Toll/IL-1 receptor family, which enables them to sense changes in their surrounding milieu. Depending on the precise extracellular stimulus, MCs respond by releasing preformed mediators from granules through a process called degranulation, and by de novo synthesis of lipid-derived mediators, cytokines and chemokines. The IL-1-type cytokine IL-33 induces the production of proinflammatory cytokines and chemokines from MCs. The first part of this study developed from an initial investigation to further detail IL-33-stimulated ERK1/2, p38 and cPLA2 signaling kinetics. We revealed that IL-33-stimulated MCs secrete factors within the first 30 min that are able to induce ERK1/2 and Akt phosphorylation in an autocrine fashion. We found that, similar to LTB4, these secreted factors triggered ERK1/2 and Akt activation in a Gi protein- and class IB PI3K-dependent manner. By employing PTX and pharmacological inhibitors targeting 5LO/FLAP, we revealed that 5LO-derived factors signaling through PTX-sensitive GPCRs play a part in IL-33-stimulated ERK1/2 signaling in bone marrow-derived MCs (BMMCs). This study also demonstrated that 5LO/FLAP activity and PTX-sensitive G proteins positively regulate IL-33-stimulated IL-6 production in BMMCs. Together, the results from the first part of this study suggest that IL-33 stimulation of MCs leads to the production of LTs by activated 5LO that regulate MC effector functions in an autocrine fashion. The second part of this project stemmed from previous work in our group recognizing the scaffold protein IQGAP1 as an interaction partner of Fc epsilon R1 in BMMCs. Through immunoprecipitation studies, we found that both IQGAP1 and one of its major binding partners, ß-catenin, are constitutively engaged in tyrosine-phosphorylated protein complexes in BMMCs. We showed that key factors required for Fc epsilon R1 signaling, namely the PTKs Lyn and Syk and the lipid phosphatase SHIP1, participate in these IQGAP1-containing protein complexes. To examine the functional relevance of IQGAP1 in antigen-stimulated MCs, we employed siRNA-mediated knockdown of IQGAP1 expression, achieving 50% reduction in IQGAP1 protein expression with this method. We found that IQGAP1 knockdown affected neither F-actin status nor ERK1/2 and Akt signaling in antigen-triggered BMMCs. However, IQGAP1 siRNA-treated BMMCs exhibited significantly attenuated antigen-stimulated degranulation and IL-6 production compared to cells treated with control siRNA, demonstrating that IQGAP1 positively regulates Fc epsilon R1 signaling inducing these effector functions. Collectively, we provide evidence that this scaffold protein participates in critical signaling complexes in MCs, acting as a positive regulator of Fc epsilon R1-mediated proinflammatory effector functions