Kalesnikoff Janet, Baur Nicole, Leitges Michael, Hughes Michael R, Damen Jacqueline E, Huber Michael, Krystal Gerald
Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, British Columbia, Canada.
J Immunol. 2002 May 1;168(9):4737-46. doi: 10.4049/jimmunol.168.9.4737.
We demonstrate in this study that IgE + Ag-induced proinflammatory cytokine production is substantially higher in Src homology-2-containing inositol 5'-phosphatase (SHIP)(-/-) than in SHIP(+/+) bone marrow-derived mast cells (BMMCs). Focusing on IL-6, we found that the repression of IL-6 mRNA and protein production in SHIP(+/+) BMMCs requires the enzymatic activity of SHIP, because SHIP(-/-) BMMCs expressing wild-type, but not phosphatase-deficient (D675G), SHIP revert the IgE + Ag-induced increase in IL-6 mRNA and protein down to levels seen in SHIP(+/+) BMMCs. Comparing the activation of various signaling pathways to determine which ones might be responsible for the elevated IL-6 production in SHIP(-/-) BMMCs, we found the phosphatidylinositol 3-kinase/protein kinase B (PKB), extracellular signal-related kinase (Erk), p38, c-Jun N-terminal kinase, and protein kinase C (PKC) pathways are all elevated in IgE + Ag-induced SHIP(-/-) cells. Moreover, inhibitor studies suggested that all these pathways play an essential role in IL-6 production. Looking downstream, we found that IgE + Ag-induced IL-6 production is dependent on the activity of NF-kappa B and that I kappa B phosphorylation/degradation and NF-kappa B translocation, DNA binding and transactivation are much higher in SHIP(-/-) BMMCs. Interestingly, using various pathway inhibitors, it appears that the phosphatidylinositol 3-kinase/PKB and PKC pathways elevate IL-6 mRNA synthesis, at least in part, by enhancing the phosphorylation of I kappa B and NF-kappa B DNA binding while the Erk and p38 pathways enhance IL-6 mRNA synthesis by increasing the transactivation potential of NF-kappa B. Taken together, our data are consistent with a model in which SHIP negatively regulates NF-kappa B activity and IL-6 synthesis by reducing IgE + Ag-induced phosphatidylinositol-3,4,5-trisphosphate levels and thus PKB, PKC, Erk, and p38 activation.
我们在本研究中证明，在含Src同源2结构域的肌醇5'-磷酸酶（SHIP）基因敲除（SHIP(-/-)）的骨髓来源肥大细胞（BMMCs）中，IgE + 抗原诱导的促炎细胞因子产生显著高于SHIP基因正常（SHIP(+/+)）的BMMCs。以白细胞介素-6（IL-6）为研究对象，我们发现SHIP(+/+) BMMCs中IL-6 mRNA和蛋白产生的抑制需要SHIP的酶活性，因为表达野生型而非磷酸酶缺陷型（D675G）SHIP的SHIP(-/-) BMMCs可使IgE + 抗原诱导的IL-6 mRNA和蛋白增加恢复到SHIP(+/+) BMMCs中的水平。通过比较各种信号通路的激活情况以确定哪些通路可能导致SHIP(-/-) BMMCs中IL-6产生增加，我们发现磷脂酰肌醇3激酶/蛋白激酶B（PKB）、细胞外信号调节激酶（Erk）、p38、c-Jun氨基末端激酶和蛋白激酶C（PKC）通路在IgE + 抗原诱导的SHIP(-/-)细胞中均被激活。此外，抑制剂研究表明所有这些通路在IL-6产生中都起重要作用。进一步研究发现，IgE + 抗原诱导的IL-6产生依赖于核因子κB（NF-κB）的活性，并且SHIP(-/-) BMMCs中IκB的磷酸化/降解以及NF-κB的易位、DNA结合和反式激活都明显更高。有趣的是，使用各种通路抑制剂后发现，磷脂酰肌醇3激酶/PKB和PKC通路至少部分通过增强IκB的磷酸化和NF-κB与DNA的结合来提高IL-6 mRNA的合成，而Erk和p38通路则通过增加NF-κB的反式激活潜能来增强IL-6 mRNA的合成。综上所述，我们的数据与这样一个模型一致，即SHIP通过降低IgE + 抗原诱导的磷脂酰肌醇-3,4,5-三磷酸水平，从而减少PKB、PKC、Erk和p38的激活，对NF-κB活性和IL-6合成起负向调节作用。
