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Regulation of mast cell activation Kalesnikoff, Janet


SH2-containing inositol 5'-phosphatase (SHIP) is a 145kDa protein that becomes both tyrosine phosphorylated and associated with the adapter protein She following the stimulation of hemopoietic cells with a variety of extracellular stimuli. SHIP typically acts as a negative regulator of hemopoietic cell activation, at least in part, by hydrolyzing the phosphatidylinositol 3'-kinase (PI3K) generated second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). To gain further insight into SHIP'S role in mast cell development and activation, we generated bone marrow-derived mast cells (BMMCs) from SHIP+/+ and -/- mice. We found that mature BMMCs from these mice exhibit comparable receptor expression profiles, total granularity and granular content, despite the faster rate of development observed in the absence of SHIP. Following stimulation of these cells, we found that IgE (Immunglobulin E) + antigen (Ag)- induced degranulation, arachidonic acid (AA) metabolism, and proinflammatory cytokine production are substantially higher in SHIP-/- than +/+ BMMCs. Focusing on cytokine production, we demonstrate herein that SHIP negatively regulates interleukin (IL)-6 production by inhibiting nuclear factor κB (NFKB) activity. Using various pathway inhibitors we determined that the PI-3K/Protein Kinase (PK)B and PKC pathways, which are elevated in IgE+Ag-induced SHIP-/- cells, elevate IL-6 mRNA synthesis, at least in part, by enhancing the phosphorylation of κB and NFκB DNA binding. Conversely, the Erk and p38 pathways, which are also elevated in SHIP-/- cells, enhance IL-6 mRNA synthesis by increasing the transactivation potential of NFκB . Taken together, our results are consistent with a model in which SHIP negatively regulates NFκB activity and IL-6 synthesis by reducing IgE+Ag-induced PIP₃ levels and thus PKB, PKC, Erk and p38 activation. Although IgE binding to mast cells is thought to be a passive pre-sensitization step in the current mast cell paradigm, we observed that SHIP-/- BMMCs degranulate in response to IgE alone, unlike their wildtype counterparts. We explored this phenomenon further and found that monomeric IgE (mlgE), in the absence of Ag, stimulates multiple phosphorylation events in normal BMMCs. While mlgE does not induce degranulation or leukotriene synthesis, it leads to a more potent production of cytokines than IgE+Ag. Moreover, mlgE prevents the apoptosis of cytokine-deprived BMMCs, likely by maintaining BcI-X[sub L] levels and inducing the production of autocrine-acting cytokines. Since IgE concentrations as low as 0.1 μg/ml enhance BMMC survival, elevated plasma IgE levels in humans with atopic disorders may contribute to the elevated mast cell numbers seen in these individuals. We also found that IgE alone triggers the adhesion of mast cells to the connective tissue component fibronectin (FN). This adhesion occurs to the same extent as that triggered by optimal levels of stem cell factor (SCF) or IgE+Ag and is mediated by an increased avidity of integrin α₅β₁. Moreover, IgE-induced adhesion requires PI3K, phospholipase C (PLC)γ and extracellular calcium (Ca²⁺) but not Erk or p38. We demonstrated, using a Ca²⁺ channel blocker and Lyn-/- BMMCs, that both IgE- and IgE+Ag-induced adhesion to FN require extracellular Ca²⁺ entry whereas SCF-induced adhesion does not. Furthermore, our data suggest that FN acts synergistically with IgE to prolong intracellular phosphorylation events and enhance IgE-induced cytokine production and BMMC survival.

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