Defining the roles of adenosine, anesthetics, and selective integrin targeting during leukocyte and platelet activation -A basis for new therapeutic strategies aiming to treat systemic inflammation, ischemia-reperfusion injury and thrombosis

全身炎症反应综合征(SIRS)是重症监护医学中发病率和死亡率的主要原因。在脓毒症期间，SIRS是由感染性微生物引起的。SIRS也可以由非感染性原因引起，如心脏手术中的体外循环(ECC)或器官缺血和再灌注。在SIRS过程中，促炎介质诱导白细胞、血小板和内皮细胞的激活和相互作用。这些细胞间的相互作用对维持SIRS和导致危及生命的炎性器官衰竭至关重要。白细胞的激活与包括Mac-1整合素受体在内的黏附分子的表达和配体亲和力的增加有关。Mac-1在白细胞与血管内皮细胞和血小板的黏附中起核心作用。相比之下，胞外脱氧核糖核酸酶CD39在产生抗炎分子腺苷方面起着关键作用，腺苷可以抑制白细胞和血小板的激活。麻醉剂七氟醚和异丙酚也有抗炎的潜力。这些药物与细胞表面分子相互作用，可能激活腺苷受体。在这项研究中，我们将分析SIRS发生的潜在机制，以期建立新的药物治疗策略。为此，我们已经确定了三种有希望的方法来增加抗炎效果，我们将对其进行测试。在体外，我们将使用生理激动剂和模拟ECC来诱导白细胞和血小板的激活。在体内，我们将使用小鼠活体显微镜模型来研究SIRS期间白细胞-血小板-内皮细胞的相互作用，并将研究小鼠心肌和肝脏缺血再灌注过程中白细胞和血小板的激活情况。我们的三种方法包括：激活特异性阻断Mac-1，激活腺苷受体，以及增加腺苷产量。首先，为了确定Mac-1在白细胞和血小板相互作用中的作用，我们将使用KO小鼠品系，并分析单链抗体片段MAN-1对白细胞和血小板相互作用的影响。MAN-1选择性地与激活的Mac-1构象结合，而不影响Mac-1在免疫系统中的整体功能。其次，我们将使用七氟醚和异丙酚来诱导腺苷受体的激活，并评估这些麻醉剂对白细胞和血小板激活的影响，并观察不同的腺苷受体KO小鼠。第三，我们将使用一种二价分子，它携带胞外apyrase CD39，并针对激活的血小板GP IIb/IIIa受体。使用这种方法，apyrase的活性可以被限制在血小板和白细胞相互作用的部位，以去除血栓前血小板激动剂ADP，并产生抗炎剂腺苷。阻止白细胞和血小板激活的药理学进展代表了SIRS治疗的新颖性，因为它代表着朝着对症治疗迈出的重要一步。

The systemic inflammatory response syndrome (SIRS) is a major cause of morbidity and mortality in intensive care medicine. During sepsis, SIRS is caused by infectious microorganisms. SIRS can also result from non-infectious causes such as extracorporeal circulation (ECC) in cardiac surgery or organ ischemia and reperfusion. During SIRS pro-inflammatory mediators induce the activation and interaction of leukocytes, platelets and endothelium. These cellular interactions are of central importance for the maintenance of SIRS and lead to life-threatening inflammatory organ failure. Leukocyte activation is associated with increases in expression and ligand affinity of adhesion molecules, including the Mac-1 integrin receptor. Mac-1 plays a central role in leukocyte adhesion to endothelium and platelets. In contrast, the ecto-apyrase CD39 plays a crucial role in generating the anti-inflammatory molecule adenosine, which inhibits leukocyte and platelet activation. The anesthetics sevoflurane and propofol also carry anti-inflammatory potential. These agents interact with cell surface molecules and probably activate adenosine receptors. In this study we will analyze mechanisms underlying the development of SIRS with the aim to establish new pharmacological treatment strategies. For this purpose, we have identified three promising approaches to increase anti-inflammatory effects, which we will test. Ex vivo we will induce leukocyte and platelet activation using physiological agonists and simulated ECC. In vivo we will employ a murine intravital microscopy model to study leukocyte-platelet-endothelium interaction during SIRS and will investigate leukocyte and platelet activation during ischemia and reperfusion of mouse myocardium and liver. Our three approaches include: Activation-specific blockade of Mac-1, activation of adenosine receptors, and increase of adenosine production. First, to define the role of Mac-1 in leukocyte and platelet interaction we will use a KO mouse strain and analyze the effect of the single chain antibody fragment MAN-1 on leukocyte and platelet interaction. MAN-1 selectively binds to the activated conformation of Mac-1 without affecting the overall function of Mac-1 in the immune system. Second, we will use sevoflurane and propofol to induce adenosine receptor activation and evaluate the effects of these anesthetics on leukocyte and platelet activation and look at different adenosine receptor KO mice. Third, we will employ a bivalent molecule, which carries the ecto-apyrase CD39 and is targeted against the activated platelet GP IIb/IIIa receptor. Using this approach, apyrase activity can be confined to sites of platelet-leukocyte interaction to remove the prothrombotic platelet agonist ADP and to produce the anti-inflammatory agent adenosine. Pharmacological advances to prevent leukocyte and platelet activation represent a novelty in the treatment of SIRS, as it represents a major step towards symptomatic treatment.