van der Bruggen T, Kanters D, Tool A T, Raaijmakers J A, Lammers J W, Verhoeven A J, Koenderman L
Department of Pulmonary Diseases, University Hospital Utrecht, The Netherlands.
J Allergy Clin Immunol. 1998 Jan;101(1 Pt 1):103-9. doi: 10.1016/S0091-6749(98)70200-3.
Human eosinophils are strongly modulated by the eosinophilotrophic cytokines IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). A clear intracellular effect of these cytokines is the induction of tyrosine phosphorylation of multiple cellular substrates. However, the relevance of tyrosine phosphorylation for eosinophil functioning has not been established.
In this study we have investigated dose-response and time curves of IL-5-, IL-3-, and GM-CSF-induced tyrosine phosphorylation in eosinophils. Moreover, we have evaluated the importance of IL-5-induced tyrosine phosphorylation for priming of human eosinophils.
Cytokine-induced tyrosine phosphorylation was monitored on western blot with an antiphosphotyrosine antibody (4G10). To probe the relevance of tyrosine phosphorylation for priming, eosinophils were primed with IL-5 in the presence of the tyrosine kinase inhibitor herbimycin A. Platelet activating factor (PAF) was used as a control priming agent. Subsequently, the eosinophils were incubated with serum-treated zymosan (STZ) to activate the respiratory burst. Binding of STZ was determined by FACS analysis.
IL-5-, IL-3-, and GM-CSF-induced tyrosine phosphorylation was found at concentrations that primed eosinophil effector mechanism (median effective dose values: approximately 5.10(-11) mol/L, approximately 5.10(-10) mol/L, and approximately 5.10(-12) mol/L for IL-5, IL-3, and GM-CSF, respectively). Cytokine-induced tyrosine phosphorylation was transient with an optimum value at 15 minutes. IL-5 priming of STZ-induced activation of the respiratory burst was blocked by herbimycin A, whereas PAF still primed this response. In fact, herbimycin A inhibited IL-5 priming of STZ binding to human eosinophils. On the other hand, PAF priming of STZ binding was not affected by herbimycin A. Both IL-5-induced and PAF-induced tyrosine phosphorylation were inhibited by herbimycin A.
These data demonstrate for the first time that IL-5 priming of opsonized particle-induced responses is mediated by tyrosine kinase activity in human eosinophils.
人嗜酸性粒细胞受到嗜酸性粒细胞营养因子细胞因子IL-5、IL-3和粒细胞-巨噬细胞集落刺激因子（GM-CSF）的强烈调节。这些细胞因子在细胞内的一个明显作用是诱导多种细胞底物的酪氨酸磷酸化。然而，酪氨酸磷酸化与嗜酸性粒细胞功能的相关性尚未明确。
在本研究中，我们研究了IL-5、IL-3和GM-CSF诱导嗜酸性粒细胞酪氨酸磷酸化的剂量反应曲线和时间曲线。此外，我们评估了IL-5诱导的酪氨酸磷酸化对人嗜酸性粒细胞致敏的重要性。
用抗磷酸酪氨酸抗体（4G10）通过蛋白质印迹法监测细胞因子诱导的酪氨酸磷酸化。为了探究酪氨酸磷酸化对致敏的相关性，在酪氨酸激酶抑制剂赫曲霉素A存在的情况下，用IL-5对嗜酸性粒细胞进行致敏。血小板活化因子（PAF）用作对照致敏剂。随后，将嗜酸性粒细胞与血清处理的酵母聚糖（STZ）孵育以激活呼吸爆发。通过流式细胞术分析测定STZ的结合。
在能够引发嗜酸性粒细胞效应机制的浓度下发现了IL-5、IL-3和GM-CSF诱导的酪氨酸磷酸化（中位数有效剂量值：IL-5、IL-3和GM-CSF分别约为5×10⁻¹¹mol/L、约5×10⁻¹⁰mol/L和约5×10⁻¹²mol/L）。细胞因子诱导的酪氨酸磷酸化是短暂的，在15分钟时达到最佳值。赫曲霉素A阻断了IL-5对STZ诱导的呼吸爆发激活的致敏作用，而PAF仍能引发这种反应。实际上，赫曲霉素A抑制了IL-5对STZ与人嗜酸性粒细胞结合的致敏作用。另一方面，PAF对STZ结合的致敏作用不受赫曲霉素A的影响。赫曲霉素A抑制了IL-5诱导的和PAF诱导的酪氨酸磷酸化。
这些数据首次证明，调理素化颗粒诱导反应的IL-5致敏作用是由人嗜酸性粒细胞中的酪氨酸激酶活性介导的。
