Supplementary Material for: Interleukin 8 Elicits Rapid Physiological Changes in Neutrophils That Are Altered by Inflammatory Conditions

A sufficient response of neutrophil granulocytes stimulated by interleukin (IL)-8 is vital during systemic inflammation, for example, in sepsis or severe trauma. Moreover, IL-8 is clinically used as biomarker of inflammatory processes. However, the effects of IL-8 on cellular key regulators of neutrophil properties such as the intracellular pH (pH_i) in dependence of ion transport proteins and during inflammation remain to be elucidated. Therefore, we investigated in detail the fundamental changes in pH_i, cellular shape, and chemotactic activity elicited by IL-8. Using flow cytometric methods, we determined that the IL-8-induced cellular activity was largely dependent on specific ion channels and transporters, such as the sodium-proton exchanger 1 (NHE1) and non-NHE1-dependent sodium flux. Exposing neutrophils in vitro to a proinflammatory micromilieu with N-formyl-Met-Leu-Phe, LPS, or IL-8 resulted in a diminished response regarding the increase in cellular size and pH. The detailed kinetics of the reduced reactivity of the neutrophil granulocytes could be illustrated in a near-real-time flow cytometric measurement. Last, the LPS-mediated impairment of the IL-8-induced response in neutrophils was confirmed in a translational, animal-free human whole blood model. Overall, we provide novel mechanistic insights for the interaction of IL-8 with neutrophil granulocytes and report in detail about its alteration during systemic inflammation.

白细胞介素8（interleukin, IL-8）刺激中性粒细胞（neutrophil granulocytes）所产生的充分应答，在全身性炎症（如脓毒症或严重创伤）过程中至关重要。此外，IL-8已被临床用作炎症进程的生物标志物。然而，IL-8对中性粒细胞特性关键细胞调控因子（如细胞内pH，intracellular pH, pHi）的影响，在依赖于离子转运蛋白的条件下以及炎症状态中，仍有待阐明。为此，我们深入探究了IL-8诱导的细胞内pH、细胞形态及趋化活性的根本性变化。借助流式细胞术（flow cytometric methods），我们证实IL-8诱导的细胞活性在很大程度上依赖于特定离子通道与转运蛋白，例如钠氢交换体1（sodium-proton exchanger 1, NHE1）以及非NHE1依赖型钠流。将中性粒细胞体外暴露于含N-甲酰基-甲硫氨酰-亮氨酰-苯丙氨酸、脂多糖（lipopolysaccharide, LPS）或IL-8的促炎微环境后，其细胞体积增大与pH升高的应答反应均出现减弱。我们通过近实时流式细胞术检测，清晰呈现了中性粒细胞反应性降低的详细动力学过程。最后，我们在一个转化性、无动物实验的人全血模型中，验证了LPS介导的中性粒细胞IL-8应答反应受损现象。综上，本研究为IL-8与中性粒细胞的相互作用提供了全新的机制性见解，并详细报道了全身性炎症过程中该相互作用的改变情况。